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CONTENTS RESEARCH ARTICLE: Simultaneous determination of hyzetimibe and its main active metabolite in plasma by LC–MS/MS and its application in PK study Bioanalysis Vol. 7 Issue 15 RESEARCH ARTICLE: Highly specific and sensitive immunoassay for the measurement of prostaglandin E2 in biological fluids Bioanalysis Vol. 7 Issue 19 REVIEW: Derivatization methods for LC–MS analysis of endogenous compounds Bioanalysis Vol. 7 Issue 19 METHODOLOGY: Development of an Excel-based laboratory information management system for improving workflow efficiencies in early ADME screening Bioanalysis Vol. 8 Issue 2 Research Article

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7 Research Article 2015/07/30 Simultaneous determination of hyzetimibe and its main active metabolite in plasma by LC–MS/MS and its application in PK study

Bioanalysis Background: Hyzetimibe is a new compound belonging to a novel class of selective Jinliang Chen1, Honggang cholesterol absorption inhibitors. A simple, highly sensitive LC–MS/MS method has Lou1, Bo Jiang1, Rong Shao1, been developed for the quantification of hyzetimibe and its main active metabolite, Zourong Ruan1 ,1 hyzetimibe-glucuronide, in human plasma. Results: Analytical samples were prepared & Jian’an Wang* 1Center of Clinical Pharmacology, using a protein precipitation method coupled with a concentration process. The the Second Affiliated Hospital of linearity of this method was established for concentrations in the ranges of 0.05–50 Zhejiang University School of Medicine, and 0.5–500 ng/ml for hyzetimibe and hyzetimibe-glucuronide, respectively. The Hangzhou, Zhejiang, China accuracy and precision of the method varied from 97.9 to 105% and 2.6 to 7.4%, *Author for correspondence: respectively. Conclusion: This study represents the first reported example of an Tel.: +86 0571 8778 3759 Fax: +86 0571 8778 3969 LC–MS/MS assay for the simultaneous quantification of hyzetimibe and its main active [email protected] metabolite, hyzetimibe-glucuronide, in human plasma. Furthermore, this method has been successfully applied to a PK study.

Hyzetimibe (1-[4-fluorophenyl]-3[R]-[3-(4- pletely converted to its active metabolite fluorophenyl)-4-hydroxybut-2(Z)-enyl]-4 hyzetimibe-glucuronide (1-O-[4-(trans- [S]-[4-hydroxyphenyl]-2-azetidinone) is a [2S,3R]-1-[4-Fluorophenyl]-3[R]-[3-(4- new cholesterol absorption inhibitor fluorophenyl)-4-hydroxybut-2(Z)-enyl]-4 that blocks the intestinal absorption of cho- [S]-[4-hydroxyphenyl]-2-azetidinyl) lesterol and phytosterols. As novel choles- phenyl]-β-d-glucuronic acid). On aver- terol-lowering agents, cholesterol absorption age, the AUC of hyzetimibe-glucuronide inhibitors can be used to significantly lower was approximately 90% of the exposure of the levels of low-density lipoprotein choles- the total amount of the compounds in the terol (LDL-C) in humans, when they are plasma (i.e., hyzetimibe plus hyzetimibe- used alone or in combination with statins [1] . glucuronide) [2]. In a similar manner to 15 The results of an initial PD study in healthy ezetimibe, significant levels of enterohepatic subjects showed that the once-daily adminis- recycling occurred following the oral admin- tration of hyzetimibe led to significant reduc- istration of hyzetimibe, which resulted in tions in LDL-C by 7.8–19.7% [2]. The results multiple peaks in the concentration–time 2015 of a similar study using the first reported profiles for this drug [4]. For this reason, cholesterol adsorption inhibitor, ezetimibe, there is an urgent need for the development revealed an 18.5% reduction in LDL-C in of a reliable-validated method for the PK subjects [3]. Hyzetimibe appears to be safe study of hyzetimibe in humans, especially and well tolerated, and the results of all of for the concentration of hyzetimibe-gluc- the preclinical studies and Phase I clinical uronide. We previously reported the first- trials to have been conducted to date suggest in-human study of hyzetimibe, where pro- that this drug could be used as a potential tein precipitation was used to allow for the treatment for hypercholesterolemia. simultaneous determination of hyzetimibe Hyzetimibe is rapidly absorbed in and hyzetimibe-glucuronide [2]. To the best the small intestine following its oral of our knowledge, there have been no other administration and is almost com- reports in the literature pertaining to the part of

Key terms has also been used to support a PK study in healthy subjects. Cholesterol absorption inhibitor: Agents that act at the brush border of the small intestine to inhibit the intestinal absorption of dietary and biliary cholesterol across the Materials & methods intestinal wall, leading to a decrease in the delivery of Chemicals & materials intestinal cholesterol to the liver. Hyzetimibe (Lot no.: 120101, 99.0%) and hyzeti- Isotope-labeled IS: Nonradioactive compounds where mibe-glucuronide (Lot no.: 20110413, 91.18%) were one or more of the atoms have been exchanged with an provided by Zhejiang Hisun Pharmaceutical Co., isotope to give an IS that can be readily identified by mass Ltd (Zhejiang, China). The isotope-labeled IS spectral analysis for determination. (hyzetimibe-d4 and hyzetimibe-glucuronide-d4) were obtained from Shanghai ChemPartner Co., systematic determination of hyzetimibe and hyzeti- Ltd (Shanghai, China). The structures of these com- mibe-glucuronide in plasma. The chemical structures pounds are shown in Figure 1. Acetonitrile and metha- of hyzetimibe and ezetimibe are very similar, and the nol were purchased as the HPLC grade from Merck methods reported in the literature for the determi- (Darmstadt, Germany). HPLC-grade water was puri- nation of ezetimibe involve the individual quantified using a Milli-Q water purification system (Milli- fication of free ezetimibe and total ezetimibe (after pore Corp., MA, USA). All of the other chemicals used hydrolysis) [5,6,7]. However, these methods could be in the current study were purchased as the analytical complicated by the hydrolysis of ezetimibe, because grade from Sinopharm Chemical Reagent Company this process could affect the final concentration. (Shanghai, China). In this study, we have developed a simple and highly sensitive LC–MS/MS method with a LLOQ, LC–MS/MS which has been combined with protein precipitation The LC system consisted of a Nexera LC (LC-30AD), and concentration processes to allow for the simulta- a Nexera column oven (CTO-30A), which was set neous quantification of the plasma concentrations of at 40°C (Shimadzu, Kyoto, Japan), and an autos- hyzetimibe and hyzetimibe-glucuronide. This method ampler (PAL HTC-xt), which was set at 4°C (CTC, Zwingen, Switzerland). The analytes were separated over a 1.8 μm Agilent ZORBAX SB-C18 column (50 × 2.1 mm; CA, USA) under gradient conditions using water (mobile phase A) and methanol (mobile phase B) as the eluents. The gradient cycle consisted of an initial 1.8 min isocratic elution with 5% mobile phase B, followed by a linear increase in mobile phase B to 95% over a period of 2 min. The column was then eluted for 0.7 min with 95% mobile phase B before being eluted with 5% mobile phase B for 1 min. The total run time for each injection was therefore 5.5 min at a flow rate of 0.3 ml/min with a backpressure of approximately 30 MPa. The LC system was coupled to a QTRAP® 5500 System (AB SCIEX, CA, USA) fitted with a Turbo V™ ionization source interface, which was operated in the negative ion mode. Quantification was performed using the multiple reaction monitoring mode to monitor the following transitions: m/z 420.0→283.0 (hyzetimibe), m/z 595.8→283.0 (hyzetimibe-gluc-

uronide), m/z 424.0→287.0 (hyzetimibe-d4) and

m/z 599.9→287.0 (hyzetimibe-glucuronide-d4). The ARE YOU IN THE ZONE? source dependent parameters, which were maintained for all analytes were as follows: curtain gas (CUR), nitrogen; flow rate, 30 l/min; collision gas (CAD), Connect and interact with medium; ionspray voltage (IS), –4500 eV; temperature (TEM), 500°C; ion source gas 1 (GS 1) flow rate, international bioanalysts 40 l/min; ion source gas 2 (GS 2) flow rate, 60 l/min.

1858Join today Bioanalysis (2015) 7(15) future science group www.Bioanalysis-Zone.com Simultaneous determination of hyzetimibe & its metabolite in plasma Research Article

A B

283.0 283.0 OH 7. 5e6 O GlGlu 2.6e8 OH 7. 0e6 OH 2.4e8 6.5e6 N 2.2e8 N F O 6.0e6 F O

2.0e8 F 5.5e6 F 1.8e8 5.0e6 1.6e8 4.5e6 1.4e8 4.0e6 1.2e8 3.5e6 112.8 3.0e6 1.0e8 2.5e6 8.0e7 2.0e6 6.0e7 1. 5e6 174.6 - 4.0e7 [M-H] 265.2 265.2 420.0 1. 0e6 [M-H]- 595.8 2.0e7 5.0e5 117. 1 390.0 128.9156.8 214.1 459.0 0.0 0.0 100 140180 220 260 300 340 380 420 100150 200 250 300 350 400 450 500 550 600 m/z (Da) m/z (Da) C D 287. 0 287. 0 D OH D 1. 20e8 3.6e7 O Glu OH D D OH D 1. 10e8 D N D 3.2e7 N 1. 00e8 F O D 2.8e7 F O 9.00e7 F F 8.00e7 2.4e7 7. 00e7 2.0e7 6.00e7

1. 6e7 112.9 5.00e7 174.9

4.00e7 1. 2e7 269.0

3.00e7 - [M-H] 8.0e6 424.0 2.00e7 [M-H]- 4.0e6 599.9 1. 00e7 268.9 128.7156.9 394.1 218.0 255.1 463.1 0.0 0.0 100 140180 220 260 300 340 380 420 100150 200 250 300 350 400 450 500 550 600 m/z (Da) m/z (Da)

Figure 1. Product ion mass spectra of deprotonated ions. Spectra obtained from (A) hyzetimibe, (B) hyzetimibe-glucuronide,

(C) hyzetimibe-d4 and (D) hyzetimibe-glucuronide-d4. The LC–MS/MS system was controlled using version 2/20, 5/50, 20/200 and 50/500 ng/ml. QC samples 1.6 of the Analyst® software (AB SCIEX, CA, USA). were prepared at four different concentrations, including 0.05/0.5 ng/ml (LLOQ QC), 0.15/1.5 ng/ml (low Standard solutions QC, LQC), 4/40 ng/ml (medium QC, MQC) and Hyzetimibe, hyzetimibe-glucuronide and the corre- 40/400 ng/ml (high QC, HQC). A working solution sponding isotope-labeled IS were dissolved in metha- of the IS (5/15 ng/ml, hyzetimibe-d4/hyzetimibe-gluc- nol to give 0.5 mg/ml stock solutions. Combined uronide-d4) was prepared by diluting the stock solu- working solutions of hyzetimibe (in the range of 0.5– tions in methanol. The stock and working solutions 500 ng/ml) and hyzetimibe-glucuronide (in the range were then stored at 4°C, while the QC samples were of 5–5000 ng/ml) were prepared by the serial dilution kept at -70°C until they were required for use. of the stock solutions in methanol. Working solutions of the calibration standards and QC samples were dried Sample preparation under a stream of nitrogen gas and then spiked with Fifty microliters of plasma was added to 200 μl of blank plasma. Calibration curve standards for hyzeti- acetonitrile for protein precipitation. After being mibe/hyzetimibe-glucuronide were made at seven dif- centrifuged at 35,000 × g for 10 min, 200 μl of the ferent concentrations, including 0.05/0.5, 0.1/1, 0.5/5, supernatant was transferred to another centrifuge tube

future science group www.future-science.com 1859 Research Article Chen, Lou, Jiang, Shao, Ruan & Wang

where it was concentrated at 10°C using a Refrigerated the ratios of the MF values of the analytes and the IS. CentriVap Concentrator (Labconco, MO, USA). The The CV of the IS-normalized MFs from the six differ- dried sample was dissolved by 100 μl of the IS work- ent sources was determined to allow for the evaluation ing solution (methanol), and the resulting mixture was of the matrix variability. The value of the CV should centrifuged at 35,000 × g for 10 min. Ten microliters be within ±15%. Recovery for the analyte and the IS of the supernatant was then collected for LC–MS/MS were calculated by comparing the mean area response analysis. All of the plasma samples were separated from of pre-extraction spiked sample (spiked before extrac-

blood using K2EDTA as an anticoagulant. tion) to that of extracts with postspiked samples (spiked after extraction) at each QC level. Validation The precision and accuracy properties of the method Our newly developed method was validated accord- were evaluated across the calibration range by the ing to the guidelines provided by the US FDA [8] and once-daily analysis of six replicate QC samples at four China Food and Drug Administration [9]. The selec- different concentrations levels (i.e., 0.05/0.5, 0.15/1.5, tivity of the method was assessed using six individual 4/40 and 40/400 ng/ml; hyzetimibe/hyzetimibe-gluc- sources of blank plasma, which were individually ana- uronide) for 5 days. The CV value was found to be lyzed and evaluated for interference. The absence of within ±15% of the nominal concentration for each of interfering materials was accepted when the response the concentrations tested except for the LLOQ, which was found to be less than 20% of the LLOQ for the was within ±20%. Furthermore, the back-calculated analyte and 5% for the IS. The carryover of the ana- standard concentrations were within 85–115% of lyte was experimentally determined by sequentially the nominal values except for the LLOQ, which was injecting the LLOQ sample, ULOQ sample and blank within 80–120%. plasma sample. The criterion for the carryover was the The dilution integrity was evaluated to ensure same as that of the selectivity. that the dilution of the samples with blank plasma The linearity of the method was determined by the did not adversely affect the accuracy or precision of analysis of six calibration curves, which contained the method. Blank plasma samples were spiked with seven non-zero concentrations, a blank sample (pro- hyzetimibe and hyzetimibe-glucuronide at concentra- cessed plasma without an analyte or the IS) and a zero tions above the ULOQ (i.e., 200 and 2000 ng/ml, sample (processed plasma with the IS). Each calibra- respectively), and the resulting mixtures were diluted tion curve was constructed according to the weighted with blank plasma to final concentrations of 40 and (1/x) linear regression of the peak area ratio of the ana- 400 ng/ml (n = 6), respectively. The CV values were lyte to the IS (Y) versus the corresponding nominal found to be within ±15% of the nominal values, and concentration of the analyte (X). The lowest and high- the accuracy was in the range of 85–115%. est standards on the calibration curves were accepted The stability profiles of hyzetimibe and hyzetimibe- as the LLOQ and ULOQ, respectively. A back con- glucuronide in human plasma, including their stabil- version was also conducted according to the European ity at room temperature, short-term stability at -20°C, Medicines Agency guidance (2011) [10], to evaluate the long-term stability at -70°C and stability during three possibility of a metabolite being converted to the par- freeze–thaw cycles were evaluated using two QC levels ent drug during the treatment process. Plasma samples (i.e., the LQC and HQC levels). The stability profiles were prepared containing hyzetimibe-glucuronide of hyzetimibe and hyzetimibe-glucuronide were also (ULOQ, without hyzetimibe), and the concentrations measured in the autosampler at 4°C over a period of of hyzetimibe in these samples were determined and 24 h after the plasma samples had been subjected to compared with hyzetimibe (LLOQ). protein precipitation process. These QC samples after Matrix effects were investigated using blank plasma extraction were kept in the autosampler for 24 h, and samples from six individual donors. Two QC samples then evaluated by a fresh calibration curve. The effect containing different concentrations of the analyte of hemolyzed plasma was also evaluated. Samples were (i.e., LQC and HQC samples) were used in this study. considered to be stable if the assay values were within For each analyte and the IS, the matrix factor (MF) the acceptable criterion of accuracy (±15%) and CV was calculated as the ratio of the peak area belonging (≤15%). to the analyte or the IS in the presence of the matrix (measured by analyzing blank plasma that had been PK analysis spiked with the analyte after the extraction process) A PK study was performed in healthy subjects (n = 18) relative to the peak area of the same substance in the using a protocol that had been approved by the Human absence of matrix (i.e., a pure solution of the analyte). Subject Research Ethics Committee of the Second The IS-normalized MF was evaluated by measuring Affiliated Hospital of the Zhejiang University School of

1860 Bioanalysis (2015) 7(15) future science group Simultaneous determination of hyzetimibe & its metabolite in plasma Research Article

Medicine, China. All of the subjects provided written m/z values of 420.0, 595.8, 424.0 and 599.9, respec- informed consent prior to participating in the study. tively. The most abundant product ions in the Q3 MS Hyzetimibe was administrated once-daily at a dose of spectra of hyzetimibe and hyzetimibe-glucuronide 30 mg for 14 days, and blood samples were collected gave m/z values of 283.0 under collision energies of in K2EDTA vacutainer collection tubes at predose and -20 and -35 V, respectively. The fragmentation behav- 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, 12, 16 and 24 iors of hyzetimibe and hyzetimibe-glucuronide were h postdose on days 1 and 14. Additional blood samples similar, with the majority of the fragment ions being were collected at 36, 48, 72, 96 and 120 h postdose generated by the cleavage of the azetidinone ring. on day 14. All of the blood samples were centrifuged In a similar manner, the product ions of hyzetimibe- at 2000 × g for 10 min at 4°C to separate the plasma. d4 and hyzetimibe-glucuronide-d4 gave m/z values of Samples of the plasma were collected and stored at 287.0 when they were analyzed under collision energies -70°C prior to being analyzed. In each analytical run, of -22 and -37 V, respectively. Multiple reaction moni- a calibration standard curve was injected at the begin- toring transitions of m/z 420.0→283.0, 595.8→283.0, ning and the QC samples were interspersed within the 424.0→287.0 and 599.9→287.0 were therefore used analysis samples. The PK parameters of hyzetimibe to analyze samples containing the analytes and IS and hyzetimibe-glucuronide were calculated using a (Figure 1). standard noncompartmental method with version 6.3 An interference peak (4.38 min) was observed in of the WinNolin software (Pharsight Corporation, our previous study in the 595.8/283.0 channel (hyzet- CA, USA). imibe-glucuronide) for a blank plasma sample that had been subjected to one-step protein precipitation Results & discussion process using methanol [2]. The area of this peak did The phenolic hydroxyl and carboxylic acid moieties not change with an increasing concentration of hyzet- present in the structures of hyzetimibe and hyzetimibe- imibe-glucuronide and disappeared when formic acid glucuronide, respectively (Figure 1), could be ionized was added to the mobile phase. Based on these obser- under the appropriate conditions to give the corre- vations, the interference peak was attributed to endog- sponding phenoxide and carboxylate anions. With this enous materials. However, the use of an acidic mobile in mind, all of the analyte and IS samples were ana- phase resulted in a significant decrease in the strength lyzed in the negative ion mode under turbo-ion-spray of the signals derived from the analytes. This decrease ionization conditions. The Q1 MS full scan spectra for in the signal strength was attributed to a decrease in hyzetimibe, hyzetimibe-glucuronide, hyzetimibe-d4 pH gradient within the spray droplet during the ion- and hyzetimibe-glucuronide-d4 predominantly con- ization process, where the presence of the weakly acidic tained the deprotonated precursor [M–H]– ions with formic acid effectively suppressed the deprotonation of

A B

3.0e4 1600 1400 2.5e4 1200 Area:Area: 77367736 3.33.311 2.0e4 1000 (0.5 nng/ml)g/ml)

1.5e4 800 Intensity Intensity 600 1.0e4 Area: 2330 (1 ng/ml) 400 5.0e3 200 3.75 0.0e0 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Time (min) Time (min)

Figure 2. Hyzetimibe-glucuronide at LLOQ level. (A) One-step protein precipitation process and (B) protein precipitation method coupled with a concentration process. The arrows indicate that new method can significantly reduce interference response. From peak area, the latter method provided higher response even at the lower concentration.

future science group www.future-science.com 1861 Research Article Chen, Lou, Jiang, Shao, Ruan & Wang

Table 1. Recovery and IS-normalized matrix factor of hyzetimibe and hyzetimibe-glucuronide (n = 6). Conc. (ng/ml) IS-normalized matrix factor (%) CV (%) Recovery (%) CV (%) Hyzetimibe 0.15 (LQC) 94.9 2.9 98.3 3.8 4 (MQC) ND 99.8 7.1 40 (HQC) 96.2 1.1 98.5 3.2 IS N/A 97.6 1.9 Hyzetimibe-glucuronide 1.5 (LQC) 102 2.3 98.2 6.4 40 (MQC) ND 102 4.3 400 (HQC) 113 1.1 95.1 3.5 IS N/A 101 4.4 HQC: High QC; LQC: Low QC; MQC: Medium QC; N/A: Not applicable; ND: Not detected.

the analytes in the negative ion mode [11] . Concentra- Compounds were separated using a gradient elution tion and reconstitution steps were therefore added to with a mobile phase consisting of water and methanol the analytical process following the protein precipita- at a flow rate of 0.3 ml/min. The retention times of tion step to eliminate any interference. Compared with hyzetimibe and hyzetimibe-glucuronide were approxi- the one-step protein precipitation process, the inclu- mately 4.16 and 3.30 min, respectively. The total run sion of concentration and reconstitution steps led to an time for each sample was 5.5 min. Blank plasma sam- increase in the intensities of the analyte signals and a ples from six different resources were analyzed and no reduction in the interference caused by the endogenous endogenous interference was detected. materials (Figure 2). Matrix effects were evaluated for the analytes and No significant responses were detected in the blank IS in the different plasma samples. The IS-normal- plasma following the injection of the ULOQ sample. ized MF data are shown in Table 1. These results The conversion rate from hyzetimibe-glucuronide to indicated that hyzetimibe-glucuronide underwent hyzetimibe during the treatment process was found ion-enhancement in the presence of the matrix, while to be 1.2%, which had no discernible impact of the hyzetimibe presented slight ion-suppression. The quantification process. IS-normalized MF can be used to efficiently evalu-

Table 2. Intra- and inter-day precision and accuracy for hyzetimibe and hyzetimibe-glucuronide. Nominal Intra-day (n = 6) Inter-day (n = 30) conc. (ng/ml) Measured conc. CV (%) Accuracy (%) Measured conc. CV (%) Accuracy (%) (mean ± SD; ng/ml) (mean ± SD; ng/ml) Hyzetimibe 0.05 0.056 ± 0.002 3.1 112 0.049 ± 0.004 7.4 98.9 0.15 0.162 ± 0.007 4.3 108 0.157 ± 0.006 4.1 105 4 3.7 ± 0.1 3.2 93.3 4.0 ± 0.2 4.4 101 40 42 ± 3 6.6 104 42 ± 1 3.5 104 40† 43 ± 1 3.1 106 Hyzetimibe-glucuronide 0.5 0.51 ± 0.04 7.5 103 0.49 ± 0.02 4.9 97.9 1.5 1.48 ± 0.06 4.1 98.8 1.55 ± 0.05 3.3 104 40 38 ± 2 4.3 94.7 41 ± 1 3.6 102 400 416 ± 25 6.0 104 420 ± 11 2.6 105 400† 435 ± 10 2.4 109 †Dilution QC sample, dilution factor is 5, which is done with 10 μl of high concentration sample (above ULOQ concentration, 200/2000 ng/ml) and 40 μl of blank plasma.

1862 Bioanalysis (2015) 7(15) future science group Simultaneous determination of hyzetimibe & its metabolite in plasma Research Article ate matrix effects during the quantification process. This method was found to be linear for hyzetimibe The IS-normalized MF values for both analytes were and hyzetimibe-glucuronide concentrations in the found to be in the range of 94.9–113.0% at two dif- ranges of 0.05–50 ng/ml and 0.5–500 ng/ml, respec- ferent QC levels (i.e., LQC and HQC). The CV tively. A correlation coefficient (r) of greater than 0.999 values of the IS-normalized MF were in the range was achieved by weighted (1/x) linear regression. This of 1.1–2.9%. These data suggested that the matrix newly developed method provided LLOQ values of effects would have very little impact on the quanti- 0.05 and 0.5 ng/ml, which were selected based on Cmax fication of the analytes. The mean recovery for both and signal-to-noise, where the signal-to-noise ratios the analytes ranged from 95.1 to 102% at all QC lev- were calculated by the Analyst 1.6 software (26.9 for els, and the recovery of the IS was 97.6% and 101%. hyzetimibe and 12.8 for hyzetimibe-glucuronide). It The recoveries of analytes and the IS were good and indicated that the method was sufficiently sensitive for reproducible. the PK analysis of these compounds in humans. The

A Hyzetimibe IS Hyzetimibe-glucuronide IS

120 9000 1600 700 100 8000 1400 600 7000 1200 500 80 6000 1000 5000 400 60 ensit y 800 4000

Int 300 600 40 3000 200 2000 400 20 100 1000 200 0 0 0 0 B 1. 0 2.0 3.0 4.0 5.0 1. 0 2.0 3.0 4.0 5.0 1. 0 2.0 3.0 4.0 5.0 1. 0 2.0 3.0 4.0 5.0

1800 9e4 4.15 8e4 4.15 3.29 1600 1400 3.29 8e4 7e4 1400 1200 7e4 6e4 1200 1000 6e4 5e4 1000 800 5e4 ensit y 800 4e4 4e4

Int 600 600 3e4 3e4 400 400 2e4 2e4 200 1e4 200 1e4 0 0e0 0 0e0 C 1. 0 2.0 3.0 4.0 5.0 1. 0 2.0 3.0 4.0 5.0 1. 0 2.0 3.0 4.0 5.0 1. 0 2.0 3.0 4.0 5.0 6e4 7e4 8e4 4.16 9e4 4.15 3.30 3.30 7e4 5e4 8e4 6e4 6e4 7e4 5e4 4e4 6e4 5e4

nsity 4e4 3e4 5e4 4e4 3e4 In te 4e4 3e4 2e4 3e4 2e4 2e4 2e4 1e4 1e4 1e4 1e4 0e0 0e0 0e0 0e0 D 1. 0 2.0 3.0 4.0 5.0 1. 0 2.0 3.0 4.0 5.0 1. 0 2.0 3.0 4.0 5.0 1. 0 2.0 3.0 4.0 5.0 1.8e6 4.0e5 1.8e5 4.17 1.6e5 4.17 3.29 3.29 1.6e6 1.6e5 1.4e5 3.5e5 1.4e6 1.4e5 1.2e5 3.0e5 1.2e6 1.2e5 1.0e5 2.5e5 nsity 1.0e6 1.0e5 2.0e5 8.0e4 8.0e5

In te 8.0e4 6.0e4 1.5e5 6.0e4 6.0e5 4.0e4 4.0e4 4.0e5 1.0e5 2.0e4 2.0e4 2.0e5 5.0e4 0.0e0 0.0e0 0.0e0 0.0e0 1. 0 2.0 3.0 4.0 5.0 1. 0 2.0 3.0 4.0 5.0 1. 0 2.0 3.0 4.0 5.0 1. 0 2.0 3.0 4.0 5.0 Time (min) Time (min) Time (min)Time (min)

Figure 3. Representative chromatograms of hyzetimibe (left) and hyzetimibe-glucuronide (right). Analytes in (A) human blank plasma, (B) an LLOQ sample, (C) an medium QC sample and (D) 2 h subject plasma sample after administration of 30 mg of hyzetimibe. The sample concentration was determined to be 5.8 and 22 ng/ml for hyzetimibe and hyzetimibe-glucuronide, respectively.

future science group www.future-science.com 1863 Research Article Chen, Lou, Jiang, Shao, Ruan & Wang

Table 3. Stability data for hyzetimibe and hyzetimibe-glucuronide in plasma (n = 6). Stability test Hyzetimibe Hyzetimibe-glucuronide Nominal conc. Measured conc. CV (%) Accuracy Nominal conc. Measured conc. CV (%) Accuracy (ng/ml) (mean ± SD; ng/ml) (%) (ng/ml) (mean ± SD; ng/ml) (%) RT(4 h) 0.15 0.164 ± 0.007 4.3 109 1.5 1.59 ± 0.10 6.0 106 40 42 ± 1 2.8 105 400 424 ± 8 1.8 106 Autosampler† 0.15 0.160 ± 0.008 4.8 107 1.5 1.48 ± 0.06 3.9 98.7 (24 h) 40 39 ± 1 3.7 96.8 400 393 ± 4 1.0 98.2 Freeze–thaw‡ 0.15 0.152 ± 0.004 2.6 101 1.5 1.50 ± 0.02 1.5 99.7 40 41 ± 1 2.4 101 400 418 ± 8 1.9 105 Hemolyzed 0.15 0.158 ± 0.006 3.7 105 1.5 1.50 ± 0.06 4.0 100 plasma 40 42 ± 1 2.3 104 400 405 ± 4 1.0 101 -20 °C 0.15 0.163 ± 0.006 3.4 109 1.5 1.62 ± 0.06 3.8 108 (7 days) 40 42 ± 2 4.6 104 400 423 ± 11 2.6 106 -70 °C 0.15 0.157 ± 0.008 5.0 105 1.5 1.54 ± 0.03 2.2 102 (6 months) 40 44 ± 1 2.2 111 400 415 ± 11 2.7 104 †Plasma samples stored in autosampler after treated with protein precipitation, and then evaluated by a fresh calibration curve. ‡After 3 freeze–thaw cycles. RT: Room temperature.

intraday precision and accuracy of our newly devel- mibe and hyzetimibe-glucuronide were stable in oped method were assessed with four different QC plasma for 4 h at room temperature, 24 h in an autos- samples (n = 6) containing different concentrations of ampler at 4°C after the protein precipitation process, hyzetimibe and hyzetimibe-glucuronide. The results of 7 days at -20°C and 6 months at -70°C. Furthermore, these analyzes showed that the intraday precision and repeated freeze–thaw cycles and the presence of hemo- accuracy of the method were in the ranges of 3.1–7.5% lyzed plasma did not have a discernible impact on the and 93.3–112%, respectively, for both hyzetimibe and stability, accuracy or precision of sample. hyzetimibe-glucuronide (Table 2). The interday preci- The validated method was applied to a multiple-dose sion and accuracy properties of our newly developed PK study (30 mg daily for 14 days). Plots showing the method were evaluated on five different days (n = 30). mean hyzetimibe and hyzetimibe-glucuronide plasma The results of these analyzes showed that the interday concentrations versus time are shown in Figure 4, precision values of the method were in the ranges of and the associated PK parameters are summarized in Table 4 3.5–7.4% and 2.6–4.9% for hyzetimibe and hyzeti- . The Cavg (average steady concentrations) of mibe-glucuronide, respectively and that the interday hyzetimibe and hyzetimibe-glucuronide were 4.25 and

accuracies were in the ranges of 98.9–105% and 97.9– 80.61 ng/ml. The AUClast (area under the plasma con- 105%, respectively (Table 2). The dilution integrity centration–time curve from zero to the last time point)

of the method was also evaluated using QC samples and t1/2 (half-life) values for hyzetimibe were found to that exceed the ULOQ concentration. The results be 167 ± 91 h·ng/ml and 14.7 ± 6.7 h, respectively. Sim-

of these experiments showed that the fivefold dilu- ilarly, the AUClast and t1/2 values for hyzetimibe-gluc- tion of samples with blank plasma had no discernible uronide were found to be 2770 ± 936 h·ng/ml and 15.8 impact of the accuracy or the precision of the method ± 8.3 h, respectively. These results therefore indicated (Table 2). Representative chromatograms of hyzetimibe that this new method was suitable for the PK study of and hyzetimibe-glucuronide in human blank plasma, hyzetimibe and hyzetimibe-glucuronide in humans. as well as the spectra of the LLOQ, MQC and 2 h sub- Considering the applicability of this new method in ject plasma samples after the administration of 30 mg other analogs, we have also successfully utilized it to of hyzetimibe are shown in Figure 3. simultaneous determinate ezetimibe and its metabolite Several special handling and storage conditions (data not shown). Compared with reported literatures, were investigated to assess the stability of hyzetimibe we think that our method is more advantageous to and hyzetimibe-glucuronide in plasma under a variety quantitate both parent and glucuronide metabolite in a of different conditions, and the results are shown in sample by using only a preparation and a LC run than Table 3. These stability results suggested that hyzeti- the alternative experimental design involving twice as

1864 Bioanalysis (2015) 7(15) future science group Simultaneous determination of hyzetimibe & its metabolite in plasma Research Article

A Hyzetimibe B Hyzetimibe 15 15 14 14 13 13 12 12 11 11 10 10 9 9 8 8 7 7 (ng/ml) 6 (ng/ml) 6 5 5 4 4

Mean (SD) plasma conc. 3 Mean (SD) plasma conc. 3 2 2 1 1 0 0 0510 15 20 25 020406080 100 120 Time (h post-dose, day 1) Time (h post-dose, day 14)

C D Hyzetimibe-glucuronide Hyzetimibe-glucuronide 600 600

500 500

400 400

300 300 (ng/ml ) (ng/ml ) 200 200 Mean (SD) plasma conc. Mean (SD) plasma conc. 100 100

0 0 0510 15 20 25 020406080 100 120 Time (h post-dose, day 1) Time (h post-dose, day 14)

Figure 4. Mean ± SD plasma concentration–time profiles. Hyzetimibe in human plasma following multiple dose hyzetimibe tables in (A) day 1 and (B) day 14. Hyzetimibe-glucuronide in human plasma following multiple dose hyzetimibe tables in (C) day 1 and (D) day 14. many operations plus an enzymatic hydrolysis step for essential for understanding the PK and PD properties each sample. This method could also be applied for the of these drugs. Selective cholesterol absorption inhibi- quantification of other analogs. tors are a new class of cholesterol-lowering agents, and ezetimibe was the first drug in this class to be approved Conclusion for use in humans and consequently has bright market We have developed and validated a simple, highly prospects. Many other analogs of this drug are currently sensitive and specific LC–MS/MS method for the undergoing clinical trials, including hyzetimibe. The simultaneous determination of hyzetimibe and hyzeti- plasma concentration of hyzetimibe and its main active mibe-glucuronide in human plasma. This method was metabolite, hyzetimibe-glucuronide, can be simultane- developed in accordance with the regulatory guidelines ously quantified using our newly developed method, provided by the FDA and CFDA, and represents the which requires a very low sample volume (50 μl). Based first systematic evaluation of a hyzetimibe assay. on the results of the validation parameters collected in the current study, it is envisaged that this simple, highly Future perspective sensitive and specific method for the simultaneous The development of new methods for the accurate quantification of hyzetimibe and hyzetimibe-glucuro- determination of drugs and their active metabolites is nide could be used to develop a better understanding of

future science group www.future-science.com 1865 Research Article Chen, Lou, Jiang, Shao, Ruan & Wang

Table 4. Mean PK parameters (steady state) of hyzetimibe and hyzetimibe-glucuronide after oral administration of 30 mg hyzetimibe (30 mg daily for 14 days) to 18 healthy subjects under fasting condition. Parameter Hyzetimibe (mean ± SD) Hyzetimibe-glucuronide (mean ± SD)

Cavg (ng/ml) 4.25 ± 1.82 81 ± 25

Cmax (ng/ml) 11 ± 5 414 ± 238

Tmax 2.47 ± 2.52 1.40 ± 0.83

AUClast (h.ng/ml) 167 ± 91 2770 ± 936

AUCinf (h.ng/ml) 173 ± 91 2828 ± 956

t1/2 (h) 14.7 ± 6.7 15.8 ± 8.3

the PK profile of hyzetimibe. This method could also No writing assistance was utilized in the production of this be applied for the quantification of other analogs. manuscript.

Financial & competing interests disclosure Ethical conduct of research The authors are grateful to the National Science and Technology The authors state that they have obtained appropriate institu- Major Project (No. 2011ZX09302-002-02) for financial support. tional review board approval or have followed the principles The authors have no other relevant affiliations or financial in- outlined in the Declaration of Helsinki for all human or animal volvement with any organization or entity with a financial inter- experimental investigations. In addition, for investigations in- est in or financial conflict with the subject matter or materials volving human subjects, informed consent has been obtained discussed in the manuscript apart from those disclosed. from the participants involved.

Executive summary Objective • To develop and validate an LC–MS/MS method for the simultaneous quantification of hyzetimibe and its main active metabolite in human plasma, and to apply this method in a PK study. Experimental • This method uses a very low sample volume (50 μl), and involves the combination of protein precipitation with concentration and reconstitution processes, as well as LC based on an isocratic elution. Results • The method was validated in accordance with the regulatory guidelines. Conclusion • This method represents the first report of the systematic evaluation of a hyzetimibe assay. This method was successfully applied to a multiple-dose PK study of hyzetimibe.

References healthy male subjects. Drug Metab. Dispos. 30(4), 430–437 Papers of special note have been highlighted as: • of interest (2002). 5 Li S, Liu G, Jia J, Li X, Yu C. Liquid chromatography- 1 Toth PP, Davidson MH. Cholesterol absorption blockade with ezetimibe. Curr. Drug Targets Cardiovasc. Haematol. negative ion electrospray tandem mass spectrometry method Disord. 5(6), 455–462 (2005). for the quantification of ezetimibe in human plasma. J. Pharm. Biomed. Anal 40(4), 987–992 (2006). 2 Ruan Z, Jiang B, Chen J et al. Pharmacokinetics, pharmacodynamics, safety, and tolerability of hyzetimibe • First LC–MS/MS method for determination of a cholesterol (HS-25) in healthy Chinese subjects. J. Clin. Pharmacol. absorption inhibitor in bio-fluid samples, using a hydrolysis 54(10), 1144–1152 (2014). process. • First-in-human study to assess the safety, PK and PD of 6 Oswald S, Scheuch E, Cascorbi I, Siegmund W. A LC–MS/MS hyzetimibe. method to quantify the novel cholesterol lowering drug ezetimibe in human serum, urine and feces in healthy subjects 3 Pandor A, Ara RM, Tumur I et al. Ezetimibe monotherapy genotyped for SLCO1B1. J. Chromatogr. B Analyt. Technol. for cholesterol lowering in 2,722 people: systematic review Biomed. Life Sci. 830(1), 143–150 (2006). and meta-analysis of randomized controlled trials. J. Intern. Med. 265(5), 568–580 (2009). 7 Bae JW, Choi CI, Lee JH, Jang CG, Chung MW, Lee SY. Effects of UDP-glucuronosyltransferase polymorphisms on the 4 Patrick JE, Kosoglou T, Stauber KL et al. Disposition of pharmacokinetics of ezetimibe in healthy subjects. Eur. J. Clin the selective cholesterol absorption inhibitor ezetimibe in Pharmacol. 67(1), 39–45 (2011).

1866 Bioanalysis (2015) 7(15) future science group Simultaneous determination of hyzetimibe & its metabolite in plasma Research Article

8 U.S. Department of Health and Human Services, Food and • CFDA guidance on bioanalytical method validation. Drug Administration Centre for Drug Evaluation and Research 10 European Medicines Agency (EMEA), Committee (CDER), Centre for Veterinary Medicine (CVM): Guidance for Medicinal Products for Human Use: Guideline on for Industry: bioanalytical method validation (2001). bioanalytical method validation (2011). www.fda.gov www.ema.europa.eu • US FDA guidance on bioanalytical method validation. 11 Hua Y, Jenke D. Increasing the sensitivity of an LC–MS 9 China Food and Drug Administration (CFDA), Center method for screening material extracts for organic extractables for Drug Evaluation (CDE): Guideline on clinical via mobile phase optimization. J. Chromatogr. Sci. 50(3), pharmacokinetic study of drugs (2005). 213–227 (2012). http://eng.sfda.gov.cn

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7 Research Article 2015/09/28 Highly specific and sensitive immunoassay

for the measurement of prostaglandin E2 in biological fluids

Bioanalysis 1,2 1 Background: Lack of specificity of anti-PGE2 antibodies is a long-standing problem. Yuan Gao , Rui Hou , Fen Liu1, Runlan Cai1, Given quite a few analogs and low PGE2 content in biological fluids, it is quite important to simultaneously meet the demands of high specificity and sensitivity.Results: Highly Lei Fang1, Cheng Peng**,2 ,1 specific anti-PGE antibodies were obtained by combined use of cationic carrier & Yun Qi* 2 1Institute of Medicinal Plant protein and Mannich reaction. The cross-reactivity values of the resultant polyclonal Development, Chinese Academy of and monoclonal antibodies against eight analogs were <14 and <5%, respectively. Medical Sciences & Peking Union Medical Furthermore, we established a highly sensitive ELISA, which could be applied to College, Beijing 100193, China 2Pharmacy College, Chengdu University direct analysis of PGE2 at the pg/ml level (LOQ = 15.6 pg/ml). Conclusion: We provide an appropriate strategy to develop a highly-specific and sensitive immunoassay for of Traditional Chinese Medicine, Chengdu 610075, China measuring low PGE content in biological samples. 2 *Author for correspondence: Tel.: +86 105 783 3225 Fax: +86 105 783 3225 2607 Prostaglandins (PGs) have numerous and As we know, specificity and sensitivity are diverse biological activities under various two important factors for an ideal immu- [email protected] **Author for correspondence: physiological and pathological conditions. noassay. Thus, the key of the first step in [email protected]

PGE2, which is a major lipid mediator that the development of a specific immunoas- regulates diverse biological processes, is one say is the production of a highly specific

of the most widely investigated PGs. Numer- anti-PGE2 antibody. However, in 1982, ous studies have suggested the biologically Dary et al. [11] reported that it is difficult

pivotal roles of PGE2 in cancer [1,2], inflam- to develop a highly specific PGEs antibody

mation and pain [3,4]. Additionally, PGE2 because PGEs are particularly unstable due is a major arachidonic acid metabolite and to the existence of a β-hydroxyketone moi- is involved in the modulation of immune ety. Dehydration at the 11-hydroxyl position responses as a negative feedback effector [5]. and the resultant formation of A and B series Currently, the methods for detecting of PGs can occur during the coupling of 19 PGE2 are predominantly based on instru- hapten to the carrier protein and by plasma mental and immunological methods. Early isomerase enzymes [12] . Thus, the resulting

studies demonstrated that PGE2 can be antibody may recognize not only PGEs but detected by reversed phase (RP)-HPLC com- also PGAs and PGBs. Tanaka et al. [13] also 2015 bined with a UV detector at 195 nm [6–8], reported that the production of specific anti-

as well as by GC [7]. Despite their precision, bodies to PGE2 is extremely difficult, and the these instrumental methods require expen- chemical and metabolic instability of PGEs sive equipment and are time-consuming due has been implicated as the source of this

to the saponification, extraction, cleanup, difficulty. Clinically, PGE1 is used to treat collection and concentration steps [9,10]. cerebrovascular disease, diabetes and male

Immunoassays using specific antibodies erectile dysfunction and PGF2α can be used offer advantages over chromatographic pro- to treat glaucoma. Therefore, it is important cedures because immunoassays are relatively to have highly specific anti-PGs antibodies faster, less expensive and portable. Thus, sev- for accurate determination of the types of eral immunological methods, such as ELISA, PGs in biological fluids that contain various part of are used extensively. structurally similar PGs (Figure 1).

Key terms cross-reactive with PGE1 (CR > 90%), and the CR value towards 9-dexy-9-methylene-PGF2α was 100%. Immunoassay: Chemical tests used to detect or quantify a Moreover, it can be difficult to identify appropriate specific substance, the analyte, typically in a blood or body fluids sample, using an immunological reaction. mimics for other haptens. A few investigators have overcome this difficulty [15,16], but the reasons for their Hapten: Unlike complete antigen with both antigenicity success are still unclear [14] . Currently, many available and immunogenicity, hapten is a class of small molecules with antigenicity but without immunogenicity which is commercial PGE2 detection kits have considerable CR obtained only by coupling with some carrier proteins. with other PGs, especially with PGAs, PGBs, PGE1 and PGE [17,18]. Mannich reaction: Familiar amino-alkylated reaction 3 in which compounds containing active hydrogen, such This paper described a feasible method to synthesize as ketones, esters, phenols, acetylenes, α-picolines, the PGE2–carrier protein conjugate based on the cat- quinaldines, among others, can be condensed with ionic carrier protein and Mannich reaction (MR). formaldehyde and an amido in weak acidity. Satisfactorily, we obtained a highly specific anti-PGE2

In fact, the lack of specificity of PGE2 antibodies is antibody after using this conjugate as the immunogen. a long-standing problem (Table 1). As we know, anti- Furthermore, utilizing the obtained monoclonal antibody specificity depends on the antigenic determinants body (mAb), we developed a highly sensitive competi- that are exposed to immune cells. The crucial step in tive ELISA (hscELISA) based on the competition reac- developing a highly specific antibody is the preparation tion in the liquid-phase and the fluorescence detection.

of an appropriate immunogen. PGE2 is a hapten (M.W. Moreover, this assay had been validated by comparison

352.47) that is only antigenic when coupled to a carrier with a commercial PGE2 ELISA kit. As expected, the

protein. Therefore, the preparation of PGE2–carrier established hscELISA could detect the PGE2 content at protein conjugates is one of the most important steps the low pg/ml level and has been applied to the analysis

in PGE2 antibody production. Different methods of of PGE2 in biological fluids.

PGE2–protein conjugate preparation produce different

antigenic determinants. Usually, PGE2 was coupled Materials & methods with carrier proteins using carbodiimide or chlorofor- Chemicals & materials mate. However, the resulting antibodies cross-reacted The mouse SP2/0 myeloma and RAW264.7 cell with some of the PGs (Table 1). One of these antibodies lines were purchased from the Cell Resource Center

even showed a higher affinity toward PGE1; its cross- of Peking Union Medical College (Beijing, China) reactivity (CR) value was 179% higher than that of and American Type Culture Collection (MD,

PGE2 [13] . Fitzpatrick et al. [14] used a stable PGE ana- USA), respectively. PGE2, PGE1 and the commer-

log (9-dexy-9-methylene-PGF2α) as a hapten mimic cial PGE2 ELISA kit (CPEK) were from Enzo Life

to elicit an anti-PGE2 antibody. However, the results Sciences (NY, USA). PGE3, A2, B1 and B2 were pur- showed that the resulting antisera were still highly chased from Cayman Chemical (CO, USA). nBSA,

H OH HO H H OH HO H OH HO

H* CH3 H3C H3C HOOC H* HOOC HOOC H* O O O PGE2 PGE1 PGE3

HO H HO H HO H H3C H3C H3C HOOC HOOC HOOC O O PGA1 O PGA2 PGB1

HO H HO H OH HO H OH H C 3 H3C H3C HOOC HOOC HOOC O OH OH PGB2 PGF1a PGF2a

Figure 1. Chemical structures of prostaglandins E1, E2, E3,A1,A2, B1, B2, F1α and F2α. *Indicates the active hydrogen. PGA: Prostaglandin A; PGB: Prostaglandin B; PGE: Prostaglandin E; PGF: Prostaglandin F.

2598 Bioanalysis (2015) 7(19) future science group Highly specific & sensitive immunoassay for the measurement of prostaglandin E2 in biological fluids Research Article

2-(N-morpholino)-ethane sulfonic acid (MES), 50% [4] [15] [19] [21] [13] [13] [13] [14] [14] [22] [20] PEG solution, compete and incomplete Freund’s adju- Ref. vant, hypoxanthine aminopterin thymidine, hypo-

α xanthine thymidine, alkaline phosphatase (AKP, 2 7.61 KU/mg) and 4-methylumbelliferyl phosphate 6.2 24 0.06 1.5 81 <0.2 2.8 0.25 0.20 1.5 <0.05 PGF (4-MUP) were obtained from Sigma-Aldrich (MO,

USA). Goat anti-mouse IgG + IgM antibody and α 1 goat anti-mouse IgG–HRP conjugate were purchased – – – – – – – – 0.09 2.9 – from Jackson ImmunoReasearch Laboratories, Inc. PGF (PA, USA). PGA , PGF and PGF were purchased 1 1α 2α from Santa Cruz Biotechnology, Inc. (CA, USA). 3 – – – – – – – – – – – Mouse monoclonal antibody isotyping test kit and PGE IgM purification kit were purchased from MorphoSys

AG Co. (Martinsried, Germany) and Thermo Scien- 1 6.7 47 19 – 30 179 18 12 90 100 14.29 tific (MA, USA), respectively. Indomethacin was from PGE TCI chemicals Co. (Shanghai, China). Other reagents

were purchased from Beijing Chemical Reagents Co. 2 (Beijing, China). CR values (%) 0.04 – 16 – 1.6 <0.2 67 0.03 0.009 0.05 0.67 PGB

Buffers & solutions 1 Ultrapure deionized water was used for the preparation – – – 70 – <0.2 – – 0.005 0.1 PGB of the following buffers and solutions: coating buffer: – 0.05 M carbonate buffer, pH 9.6; assay buffer: 10 mM

2 Tris–HCl with 4 mM MgCl2 and 0.2 mM ZnCl2, 0.26 – 100 – 5.1 <0.2 5.6 PGA 0.17 0.75 2.8 pH 8.0; washing buffer: assay buffer with 0.05% 2.86 (v/v) Tween 20; blocking solution: 5% goat serum in assay buffer; conjugation buffer: 0.1 M MES, pH 1 † 0.24 – 53.3 – 0.9 <0.2 – 10.0 0.08 4.9 0.67 4.75; 4-MUP substrate solution: 0.3 mg/ml 4-MUP in PGA

1.0 mM MgCl2 and 0.1 M diethanol amine, pH 9.8; pNPP substrate solution: 10 mM pNPP in 1.0 mM

MgCl2 and 0.1 M diethanol amine, pH 9.8; stop solution: 0.5 M NaOH. antibodies in representative literatures. Cationization 2 mAb mAb pAb mAb mAb (29/11) mAb (195/69) mAb pAb pAb pAb Type of antibodyType pAb The preparation of cationic bovine serum albumin (cBSA) (Figure 2 steps 1–5) followed our previously described method [23].

Preparation of PGE2–protein conjugates

Preparation of PGE2-nBSA & PGE2–cBSA conjugates

The preparation of PGE2–protein conjugates with a MR was performed as previously described (Figure 2 – Carbodiimide Carbodiimide Carbodiimide Ethylchloroformate Ethylchloroformate Isobutyl chloroformate Ethylchloroformate EDC EDC Coupling reagent reagent Coupling Isobutyl chlorocarbonate steps 6–9) [24], with some modifications. Briefly, 1.0 mg of protein (native bovine serum albumin [nBSA] or cBSA) and 250 μg of PGE2 were dissolved in 50 μl of conjugation buffer and 250 μl of N, N-dimethylfor- Prostaglandin F. mamide, respectively. After dropwise addition of the protein solution into the PGE2 solution, the mixture was shook gently. A total of 60 μl of 37% formaldehyde was added to the mixture, and the mixture was )-KLH )-BSA -globulin γ α α -KLH -thyroglobulin -oxime-BSA -thyroglobulin - -BSA -BSA shook gently for 12 h at 37°C. After centrifugation, the -bovine thyroglobulin -BSA 2 2 2 2 2 2 2 2 2 supernatant was dialyzed exhaustively for 72 h until it Prostaglandin E; PGF: Table 1. Cross-reactivity 1. Table of reported anti-prostaglandin E Not mentioned in literature PGE † PGE PGE PGE PGE PGE (9-dexy-9-methylene- PGF2 (9-dexy-9-methylene- PGF2 PGE PGE PGE Immunogen EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; KLH: Keyhole limpet hemocyanin; mAb: Monoclonal antibody; pAb:PGE: Polyclonal antibody; PGA: Prostaglandin A; PGB: Prostaglandin B; was lyophilized.

future science group www.future-science.com 2599 Research Article Gao, Hou, Liu et al.

H HCl N+ N N H Ö H H OH HO H C NH C 2 O * H H H Protein H C H Protein 3 (3)+ H H N H* (2) O N C NH2 HOOC C HO NH H H H 2 O H2N (6) +H+ (4) + Cl- NH (1) N HO H OH NH H 2 C * H H C H H Protein 3 H* H + N N HOOC Ö N C O H O+ H H (7) + (5) -H -H2O NH2 NH Protein HO H OH 2 HO H C H Protein H C 3 H N O H C +N C HOOC H H O H Ö H

(8) NH NH H OH 2 H OH 2 HO H HO H H Protein H Protein H C N H C N (9) 3 N C 3 H N C HOOC HOOC H -H+ O O H H + O O H

Figure 2. A schematic diagram based on position 10-H for the preparation of prostaglandin E2–cationic protein conjugates. (1 and 2) show the cationization of protein; (3–5) show the imidization of N-hydroxymethyl amine groups in the cationic protein;

(6 and 7) show the enolation of prostaglandin E (PGE)2; and (8 and 9) show the conjugation of enolated PGE2 with the iminium ionized protein. All of the reactions were carried out in MES buffer. This reaction can be proceeded in other directions, such as with the active hydrogens on positions 2 and 8. The PGE2 conjugates with cationic protein reactions may be a mixture of conjugates with different conjugation sites.

Preparation of PGE2–AKP conjugates 2,4,6-trinitrobenzene 1-sulfonic acid (TNBS) assay It is important to note that the MR may not be suitable as previously described [26], with slight modifications. to prepare enzyme conjugates for inactivation under Briefly, 1.0 ml of the protein conjugate solution was

organic conditions. Here, we chose a single-step glu- added to 1.0 ml of 4% NaHCO3 (pH 8.5) and 1.0 ml of taraldehyde method [25]. Briefly, after dropwise addi- 0.05% freshly prepared TNBS. The reaction was carried tion of 1.0 mg (0.8 mg/ml in PBS, pH 7.2) of AKP into out at 42 ± 2°C for 2 h and was followed by the addition

the 0.1 mg (5 mg/ml in acetone) PGE2 solution, the of 1.0 ml 10% sodium dodecyl sulfate and 0.5 ml of 1.0 mixture was dialyzed against 450 ml PBS (pH 7.2) at M HCl. The absorbance of the solution was monitored 4°C for 24 h. The reaction proceeded at room tempera- at 335 nm. The number of e-amino groups present in

ture for 2 h after the addition of 100 μl of 2.5% (v/v, the carrier protein/PGE2–protein conjugates was directly dissolve in PBS, pH 7.2) glutaraldehyde. After dialysis determined from a standard curve generated from the in 1000 ml assay buffer at 4°C for 12 h, the reaction difference in the absorbance at 335 nm of TNP- l -lysine

mixture (PGE2–AKP conjugate) was lyophilized and and TNP- l -glutamic acid. The difference in absorption stored at -20°C. accurately accounts for the free e-amino groups.

Calculation of conjugate molar ratio Production of pAb & mAb

The molar ratios of the conjugates of PGE2 to protein The preparation of pAb and mAb was performed (nBSA, cBSA and AKP) were calculated by using the using routine methods as we previously described [23].

2600 Bioanalysis (2015) 7(19) future science group Highly specific & sensitive immunoassay for the measurement of prostaglandin E2 in biological fluids Research Article

The PGE2-cBSA and PGE2-nBSA were used as Development & validation of the hscELISA with immunogens to immunize mice. The titers of the fluorescence detection pAb and mAb were evaluated using an indirect com- A total of 96-well microtiter plates were coated with petitive ELISA as we previously described except for 100 μl of 2.5 μg/ml goat anti-mouse IgG + IgM anti- the substitution of PGE2 for ZEN [23]. The isotype body (dissolved in the coating buffer) and were incu- of the mAb was identified by the mouse mAb iso- bated at 4°C overnight. After three washes in washing typing test kit. The ascites was pretreated by silica buffer, the plates were blocked with 200 μl of block- powder and further purified by an IgM purification ing solution for 2 h at 37°C. After washing, 100 μl kit according to the manufacturer’s instructions. All of PGE2 (or other competitors) at the different con- animal experiments were carried out according to centrations and 50 μl of 1.5 μg/ml PGE2–AKP con- the National Institutes of Health Guide for Care and jugate were added simultaneously, and then 50 μl of

Use of Laboratory Animals and approved by the Ani- 15 mg/ml purified anti-PGE2 mAb was pipetted into mals Ethics Committee of the IMPLAD of Chinese each well. Incubate the plate at 4°C for 24 h. After Academy of Medical Sciences. washing five times, 200 μl of the 4-MUP substrate solution was added. After incubation at 37 °C for 1 Development of a conventional competitive h, the reaction was terminated by 100 μl of the stop

ELISA with colorimetric method solution, and the fluorescence intensity was read atλ ex

A total of 96-well microtiter plates were coated with 355 nm and λem 460 nm.

100 μl of 12.0 μg/ml purified anti-PGE2 mAb in coat- The recovery, repeatability and the LOQ of the ing buffer and incubated at 4°C for overnight. After hscELISA based on the mAb were measured. For intra- three washes, the plates were blocked with 200 μl of assay (within-plate) repeatability, six replicates of each the blocking solution for 2 h at 37°C. After washing, dilution were tested in the same plate on a single day.

100 μl of PGE2 (or other competitors) at the different For inter-assay (between-run) repeatability, triplicates concentrations and 50 μl of 1.5 μg/ml PGE2–AKP of each dilution were run on five different days. The conjugate were added simultaneously, and the plate LOQ was calculated from the PGE2 standard curve. was incubated for 1.5 h at 37°C. After washing five To further validate the specificity of the anti-PGE2 times, 200 μl of pNPP substrate solution was added. mAb prepared in our laboratory, we purchased the After incubation at 37°C for 1 h, the enzyme reac- CPEK as a control. The spiked samples were artificially tion was stopped by the addition of 100 μl of 0.5 M spiked with eight analogs (each at final concentration

Na2CO3, and the optical density was read at 405 nm. of 100 pg/ml) and also PGE2 (at different final concentration 50, 100 and 200 pg/ml) in assay buffer. Each

Evaluation of the specificities of anti-PGE2 sample was analyzed by the hscELISA based on our antibodies anti-PGE2 mAb and the CPEK simultaneously. The

The specificity of anti-PGE2 antibodies was inves- procedure of the CPEK was carried out according to its tigated using conventional competitive ELISA directions. The accuracies of these two methods were

(ccELISA) to measure the CR values with PGE1, E3, ascertained by analysis of the recovery of PGE2. The

A1, A2, B1, B2, F1α and F2α. The antibody specificity recovery rate was calculated as follows: assay was performed by adding various free competitors at different concentrations to estimate their Recovery (%) = (amount of PGE2 in spiked respective IC50 values. Results were expressed as the sample – amount of PGE2 in unspiked sample)/added percentage inhibition as follows: amount of PGE2 × 100

% inhibition = % B/B0 Preparation of biological fluids Taking into consideration the matrix effect from where B is the optical density (OD) value at each the biological fluids, we used the hscELISA to deter- concentration of PGE2 or analyte and B0 is the OD mine PGE2 levels in the Balb/c mice sera and the value in the absence of PGE2 or analyte. The CR (%) culture supernatants from RAW 264.7 cells. For the was calculated as follows: serum assay, experimental endotoxemia was provoked via intavenous injection of lipopolysaacharide (LPS)

CR (%) = (IC50, PGE2/IC50, analyte) × 100 (10 mg/kg) in mice. Indomethacin was introduced/i.p. Key term The IC50 value was determined by using the concentration of the inhibitor that led to a 50% decrease in Matrix effects: Undesired effects of other components on the maximum signal. the measurement of the target analyte.

future science group www.future-science.com 2601 Research Article Gao, Hou, Liu et al.

Key term spectra of the hapten and the carrier protein. However, the accuracy of UV absorption methods was limited in Cationic protein: Cationized form of carrier protein our study because there was an overlap of the absorp- produced by substituting anionic side chain carboxylic groups with aminoethylamide groups. As an antigen, tion spectra of nBSA or cBSA and the PGE2. In our

the cationic protein can increase the affinity for antigen- previous experiments, a commercial PGE2 ELISA kit presenting cell membranes due to electrostatic interactions was also used to calculate the amount of PGE2 in conju- with anionic membrane phospholipids. gates; however, the results, which were inaccurate, were attributed to the steric hindrance of carrier protein. To

(20 mg/kg) 1 h prior to LPS challenge. Three hours accurately calculate the molar ratios of PGE2 and the later, the mice were anesthetized by diethyl ether. carrier protein, a sensitive and direct TNBS method

The blood samples were obtained and stored at 4°C was used in this study to analyze PGE2-nBSA, PGE2-

overnight. The sera were collected and stored at -70°C cBSA and PGE2-AKP [26]. Various hapten–protein for further use. For the culture supernatant assay, conjugates and enzyme conjugates ratios can be ana- RAW 264.7 cells were pretreated with indomethacin lyzed using the TNBS method. However, this method (10 μM) for 2 h and then stimulated with LPS (10 μg/l) cannot be used to analyze haptens that contain reactive

for 24 h. PGE2 concentrations in the serum and the primary amino groups, which can react with TNBS.

culture supernatants were simultaneously assayed by The molar ratios of PGE2-nBSA, PGE2-cBSA and

hscELISA and CPEK. At least 10- and 50-fold dilu- PGE2–AKP conjugates were determined to be 12:1, tions are needed for the culture supernatant and sera 14:1 and 4:1, respectively. tests, respectively.

Production and evaluation of anti-PGE2 Data analysis antibodies

The PGE2 standard curve was modeled as 4-Param- PGE2-cBSA and PGE2-nBSA were used as immu- eter logistic curve using the Origin_Pro_8.0. The sig- nogens and were injected (i.p.) into mice. Forty days nificance of correlation coefficient (r) was analyzed by later, the titer of the polyclonal antiserum from indi- t-test. A Student’s paired two-tailed t-test was used for vidual animals was high. Pre-immune serum controls the analysis of the difference between the measured had negligible absorbance, whereas the pAb developed

values of hscELISA and CPEK. One-way ANOVA was from both PGE2-nBSA (pAb-native) and PGE2-cBSA used to determine the statistical significance between (pAb-cationic) had a titer of 4000. different experimental groups. A Student’s unpaired Because of the high specificity of the pAb-cationic,

two-tailed t-test was used when only two groups were we produced mAb based on the PGE2-cBSA antigen. compared. Only results with p < 0.05 were considered Fifteen days after cell fusion, culture supernatants statistically significant. from each clone were screened. Positive hybridomas were subcloned three times by limiting dilution. The Results isotype of the selected mAb was IgM with a κ-type Characterization of complete antigen light chain, and the titer of ascite was 8000. After a

& PGE2- AKP purification procedure, the purity of the obtained mAb In our previous study, cationic BSA has been identified was >95%. by a UV detector and native-polyacrylamide gel elec-

trophoresis [23]. In the cBSA UV spectra, the charac- Cross-reactions of anti-PGE2 antibodies teristic absorbing wavelength was slightly blue-shifted The specificity of the antibodies was estimated by from 278 to 276 nm relative to nBSA. This shift could measuring inhibition curves using eight structurally have been caused by the increase in cBSA polarity related PG analogs. Table 2 showed that the pAb-

that is caused by excessive cationic groups and amino- native had high CR toward PGE3, with a CR value of ethylamine groups in nBSA [27]. The protein did not 79.32%, whereas the CR value for the pAb-cationic denature during native-polyacrylamide gel electropho- was 13.14%. All of the CR values of mAb with the resis, indicating that the positive charge of cationic eight analogs were satisfactory (<5%). protein can be maintained under these conditions. When the electrode was reversed, cBSA showed a Optimization & validation of the hscELISA series of bands, whereas nBSA did not migrate. Our based on the mAb previously obtained results indicated that nBSA was Owing to the low sensitivity of ccELISA (the LOQ is successfully cationized [23]. 625 ng/ml), we developed hscELISA with fluorescence Commonly, the hapten-to-carrier protein ratio is detection. Satisfactorily, the LOQ of the hscELISA determined based on the differential UV absorption based on the mAb was 15.6 pg/ml, indicating a high

2602 Bioanalysis (2015) 7(19) future science group Highly specific & sensitive immunoassay for the measurement of prostaglandin E2 in biological fluids Research Article

Table 2. Percentage of cross-reactivity with prostaglandin E2 and its eight analogs based on the conventional competitive ELISA (n = 3). Compound CR (%)† pAb-native‡ pAb-cationic§ mAb¶

PGE2 100 100 100

PGE1 34.78 < 0.1 < 0.1

PGE3 79.32 13.14 4.95

PGA1 2.01 1.89 0.73

PGA2 1.86 0.87 0.67

PGB1 7.02 4.98 4.78

PGB2 < 0.1 < 0.1 1.12

PGF1α 4.54 2.45 1.63

PGF2α 1.46 1.96 < 0.1

† The percentage of cross-reactivity is defined as the ratio of the IC50 value for prostaglandin E (PGE)2 to the IC50 relative to its competitors. ‡ Polyclonal antisera developed from PGE2–native BSA conjugate. § Polyclonal antisera developed from PGE2–cationic BSA conjugate. ¶ Monoclonal antisera developed from PGE2–cationic BSA conjugate. CR: Cross reactivity; mAb: Monoclonal antibody; pAb: Polyclonal antibody; PGA: Prostaglandin A; PGB: Prostaglandin B; PGE: Prostaglandin E; PGF: Prostaglandin F.

sensitive ELISA was established. Furthermore, opti- CR values with PGE1 and PGE3 were 70%, and 16.3%, mal assay conditions of hscELISA were determined respectively. The hscELISA and CPEK were used to by adjusting several parameters, including the con- determine the PGE2 in spiked samples simultaneously. centration of the coating antibody and the dilution of As shown in Table 5, for all spiked level, the average the PGE2–AKP conjugate. In this study, the optimal recovery rates of hscELISA were all in the range of coating concentration of the goat anti-mouse IgG + 80–120%, and the coefficients of variation were all less IgM antibody was 2.5 μg/ml, and the best dilution for than 5%. But all of the average recovery rates of CPEK the PGE2–AKP conjugate was 1:10. Using the stan- were out of the acceptable range. When we tested the Supplementary Figure 1 dard curve shown in , PGE2 PGE2 standard solution in the absence of 8 analogs, the was quantified between 15.6 and 1000 pg/ml. Under average recovery rates of these two methods were all optimal conditions, the repeatability test (Table 3) was in the range of 80% - 120%, and the CV values were (Table 4 & Supplementary Table 1) performed by comparing the% B/B0 of six replicates all less than 5% . in the same plate (intra-assay repeatability) or those of It is suggested that both of these two methods could triplicates on five different days (inter-assay repeatabil- accurately test PGE2 content, but the CPEK could not ity). The intra-assay CV values ranged from 0.62 to effectively distinguish PGE2 and its analogs, indicat-

4.14%, with a median value of 2.05%. The inter-assay ing that the hscELISA based on the anti-PGE2 mAb CV values were between 4.75 and 10.22%, with a in our study showed higher specific and more accurate median value of 7.40%. These data show that the assay than the commercial ELISA kit. is repeatable because it yielded low and acceptable variation. Assessment of biological fluids

When we tested the PGE2 standard solution in assay For the biological fluids assay, we chose 24 biological buffer in the absence of the eight analogs, the aver- samples (15 sera from mice and nine culture super- age recovery rates of the hscELISA based on mAb were natants from macrophages) to determine their PGE2 between 80 and 120%, and the CV values were all levels using the hscELISA and CPEK. The obtained less than 6% (Table 4). Thus, we determined that the results showed that the results of hscELISA were highly hscELISA established based on mAb could accurately correlated with that of CPEK (r = 0.967, p < 0.01). assess PGE2 content. To our knowledge, many PGE2 analogs might In order to further validate the specificity of the anti- appear in serum [11,28] or macrophage culture super-

PGE2 mAb prepared by cationic conjugate, we pur- natant [29,30]. It is because of these analogs that the chased a CPEK which served as a control. According measured values from CPEK were all higher than that to its direction, the antibody utilized in the CPEK is from hscELISA. Moreover, the differences were sig- specific for PGE2 and its closely related structures. The nificant (Student’s paired two-tailedt -test), no mat-

future science group www.future-science.com 2603 Research Article Gao, Hou, Liu et al.

Table 3. Intra- and inter-assay variations of the highly sensitive competitive ELISA.

† ‡ Prostaglandin E2 level Intra-assay Inter-assay (pg/ml) n % B/B0 (mean ± SD) CV (%) n % B/B0 (mean ± SD) CV (%) 50 6 88.9 ± 0.55 0.62 3 81.7 ± 3.88 4.75 100 6 69.1 ± 0.97 1.40 3 66.7 ± 6.82 10.22 200 6 43.5 ± 1.80 4.14 3 38.2 ± 2.77 7.24 †Intra-assay variation was calculated from six replicates on a single day. ‡Inter-assay variation was calculated from triplicates on five different days. ter sera (p < 0.01) or supernatants (p < 0.05). Notably, ketones, esters, acetylenes, etc., can be linked to amine the concentration differences between CPEK versus or amide groups by formaldehyde in weakly acidic con- hscELISA in the sera (6285 ± 3283 pg/ml; n = 15) were ditions. Compared with traditional complete antigen dramatically higher (p < 0.01) than that in the super- preparation methods, the MR has the advantages of natants (955 ± 1155 pg/ml; n = 9), indicating that there convenient operation, a short reaction time and mild

are more PGE2 analogs in the serum, rather than the reaction conditions [27], which help maintain the struc- supernatant. ture of the reactants. As shown in Table 1, previously

In addition, we further validated the hscELISA in developed anti-PGE2 antibodies based on non-MRs

popular models of LPS-induced PGE2 productions usually have high CR against PGA1, A2, B1, B2 and F2α, (in vivo and in vitro). Indomethacin, a nonselective with CR values of 53.3 [19], 100 [19], 70 [20], 67 [13] and COX inhibitor, was served as a positive control. Not 81% [13], respectively. However, the pAb-native based unexpectedly, LPS stimulation resulted in a marked on the MR had higher specificity (all of the CR values

increase in PGE2 in the macrophages culture superna- were <7.0%) suggesting that use of the MR may have

tants or the mice sera (p < 0.01). Pretreatment with improved the specificity of the anti-PGE2 antibodies

indomethacin could potently decrease PGE2 contents against PGA1, A2, B1, B2 and F2α. These results sug- (p < 0.01), even lower than the normal level, which was gest that the in vitro stage is critical for an antibody in line with previous reports (Table 6) [31,32]. development to decreasing CR to these five PGs. During the in vivo process, the complete antigen Discussion should be recognized by the antigen-presenting cells as Due to quite a few analogs (Figure 1) and low content quickly as possible before the antigen is metabolized. in biological fluids [33], specificity and sensitivity are The cationic protein was an efficient carrier and had

very important for the immunoassay of PGE2. How- many good immunological properties [24,34]. As an ever, it is quite difficult to meet these demands simul- antigen, the cationic proteins are different from native taneously. In this study, to obtain a highly specific proteins [35,36] because it can increase the affinity for

anti-PGE2 antibody, we produced the mAb based on antigen-presenting cell membranes due to electro- the MR and cationic carrier protein. To our knowl- static interactions with anionic membrane phospholip- edge, there are two key steps in the antibody produc- ids [37]. The pAb-cationic had lower CR values com- tion process that impact the specificity of anti-hapten pared with the pAb-native (Table 2). The CR values

antibodies. The first step is thein vitro stage of com- of the pAb-cationic toward PGE1 and PGE3 were <0.1 plete antigen preparation, and the second step is the in and 12.30%, respectively, whereas the CR values of vivo process after immunization. During the in vitro the pAb-native were 32.66 and 80.82%, respectively. stage, a perfect preparation of the conjugate not only These findings indicate that, as a carrier protein, cBSA

can minimize structural changes of the hapten, but plays a crucial role in decreasing the CR values of PGE1

also can fully expose the antigenic determinant. The and PGE3. Taking into account the high CR values Table 1 MR is a common amino-alkylated reaction in which with PGE1 shown in , we infer that the in vivo

compounds containing active hydrogen, such as process is critical to decreasing CR to PGE1.

Table 4. Accuracy of prostaglandin E2 determined by the highly sensitive competitive ELISA in spiked assay buffer (n = 6).

Prostaglandin E2 level (pg/ml) Measured amount (mean pg/ml ± SD) CV (%) Recovery (%) 50 53.6 ± 1.26 2.34 107.2 100 93.7 ± 3.11 3.32 93.7 200 192.0 ± 11.08 5.77 96.0

2604 Bioanalysis (2015) 7(19) future science group Highly specific & sensitive immunoassay for the measurement of prostaglandin E2 in biological fluids Research Article

Table 5. Recovery rates of prostaglandin E2 in spiked samples (n = 3).

Spiked prostaglandin E2 Detected PGE2 (pg/ml) Mean value Recovery (%) CV (%) level (pg/ml) hscELISA 50 58.90, 60.00, 58.38 59.10 118.19 1.40 100 108.83, 106.57, 106.93 107.44 107.44 1.13 200 233.06, 236.70, 239.51 236.42 118.21 1.37 CPEK† 50 188.27, 209.87, 202.16 200.10 400.21 5.47 100 233.02, 240.74, 225.31 233.02 233.02 3.31 200 287.23, 335.11, 356.38 326.24 163.12 10.86

† The commercial prostaglandin E2 ELISA kit. hscELISA: Highly sensitive competitive ELISA.

After successfully obtaining a highly specific anti- vitro requirements for complete antigen preparation. (Table 2) PGE2 mAb , we next investigated how to The cationic carrier protein satisfies thein vivo process increase the sensitivity of the immunoassay. There are requirements by having the immunological character- two improvements to be involved in our design based istics of a complete antigen. Furthermore, based on on the ccELISA. Firstly, to avoid the steric-hindrance the fluorescence detection and the adequate competi- effect and insure a more adequate competition reaction, tion in liquid-phase, we newly developed the hscELISA the reaction conditions were changed from 37°C (1.5 utilizing the obtained specific mAb, which allowed the h) in the solid-phase to 4°C (24 h) in the liquid-phase. direct specifically determining PGE2 in biological flu- As a result, it was approximately 320 times (625/1.95) ids at the low pg/ml level. Collectively, our study pro- more sensitive, based on the LOQ (1.95 ng/ml), than vides an appropriate strategy to develop a highly spe- the ccELISA (LOQ = 625 ng/ml). Secondly, taking cific and sensitive immunoassay for the measurement into consideration the highly sensitivity of a fluores- of low PGE2 content in biological samples. cence detection, we used a fluorescent substrate of AKP (4-MUP) instead of the colorimetric one (pNPP). As Future perspective expected, the sensitivity was further increased 125 Sensitivity and specificity are two important factors times (1.95/0.0156). Therefore, in comparison with for an excellent immunoassay. In contrast to the sen- ccELISA, the sensitivity of hscELISA was increased sitivity of an antibody, the alternative approaches for approximately 40,000 times. Moreover, the high sen- improving the specificity are few. Take the commercial sitivity can afford up to 10- to 50-fold dilutions of the PGE2 ELISA kit as an example, we can purchase a high biological samples to eliminate matrix interference and sensitivity kit, but with an unsatisfactory specificity. In thus increase the assay accuracy [38]. Taken together, fact, it is quite difficult to find a commercial kit with our established hscELISA could be applied to the direct high-specificity and sensitivity simultaneously. Herein, analysis of PGE2 in biological fluids at the pg/ml level. we developed a highly specific and sensitive hscELISA

which could be used for specifically determining PGE2 Conclusion in biological fluids at the low pg/ml level. We have The combined use of a cationic carrier protein and the a good reason to believe that the hscELISA will be MR is a viable approach to increase the specificity of extensively applied in the coming years. an antibody against PGE2. The short reaction time Besides PGE2, our strategy could be universal for and mild reaction conditions of the MR satisfy the in improving the specificity of antibodies toward other

Table 6. Analysis of the mice sera and the macrophages culture supernatants prostaglandin E2 levels by the highly sensitive competitive ELISA.

Group PGE2 in mice sera (mean ng/ml ± SD; PGE2 in culture supernatants (mean n = 5) ng/ml ± SD; n = 3) Normal control 5.61 ± 0.44 1.16 ± 0.11 LPS 15.98 ± 2.14† 9.56 ± 0.72† Indomethacin + LPS 2.41 ± 0.40‡ 0.29 ± 0.01‡ †p < 0.01 vs normal control group. ‡p < 0.01 vs LPS group. LPS: Lipopolysaacharide; PGE: Prostaglandin E.

future science group www.future-science.com 2605 Research Article Gao, Hou, Liu et al.

haptens with active hydrogens, including the analogs and 7142112), the National S&T Major Project and Scientific Researchers Aiding Enterprise Item from the Ministry of Sci- of PGE2. Furthermore, although we have demonstrated that the MR satisfies thein vitro requirements ence and Technology of the People’s Republic of China (Nos. for preparing a complete antigen and the cationic car- 2012ZX09301-002-001 and 2014ZX09201022-006) and rier protein satisfies thein vivo process requirements, it the Open Research Fund of State Key Laboratory Breeding is not mandatory to combined use of MR and cationic Base of Systematic Research, Development and Utilization of carrier protein. Researchers could reasonably select one Chinese Medicine Resources (Chengdu, P. R. China). The au- of them to improve antibody’s specificity according to thors have no other relevant affiliations or financial involve- the actual situation. Our study holds great promise for ment with any organization or entity with a financial interest developing a highly specific and sensitive immunoassay in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. of PGE2 clinically, subsequently further promoting the development of bioanalytical field. No writing assistance was utilized in the production of this manuscript. Supplementary data To view the supplementary data that accompany this paper Ethical conduct of research please visit the journal website at: www.future-science/doi/ The authors state that they have obtained appropriate institu- full/10.4155/bio.15.130 tional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal Financial & competing interests disclosure experimental investigations. In addition, for investigations in- This work was supported by the National and Beijing Natu- volving human subjects, informed consent has been obtained ral Science Foundation of China (Grant numbers 81274163 from the participants involved.

Executive summary

• Highly specific anti-prostaglandin E2 (PGE2) monoclonal antibodies were obtained by combined use of cationic carrier protein and Mannich reaction. • The sensitivity of ELISA was significantly improved by fluorescence detection and adequate competition in liquid phase.

• A highly specific and sensitive ELISA for detecting PGE2 was developed based on the resulting antibody.

• Taking into consideration the matrix effect from the biological fluids, we used the hscELISA to determine PGE2 levels in the Balb/c mice sera and the culture supernatants from RAW 264.7 cells.

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7 Review 2015/09/28 Derivatization methods for LC–MS analysis of endogenous compounds

Bioanalysis Sensitive and reliable analysis of endogenous compounds is critically important Yunting Zhu‡,1, Pan Deng‡,1 for many physiological and pathological studies. Methods based on LC–MS have & Dafang Zhong*,1 progressed to become the method of choice for analyzing endogenous compounds. 1Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike However, the analysis can be challenging due to various factors, including inherent Road, Shanghai 201203, P R China low concentrations in biological samples, low ionization efficiency, undesirable *Author for correspondence: chromatographic behavior and interferences of complex biological. The integration Tel.: +86 21 5080 0738 of chemical derivatization with LC–MS could enhance its capabilities in sensitivity and Fax: +86 21 5080 0738 selectivity, and extend its application to a wider range of analytes. In this article, we [email protected] ‡Authors contributed equally will review the derivatization strategies in the LC–MS analysis of various endogenous compounds, and provide applications highlighting the impact of these important techniques in the evaluation of pathological events. 2581 Endogenous compounds are natural and biochemical structures of analytes with diverse active substances existed in body fluids and tags [9]. The most frequently used incorpo- generated via in vivo metabolism. Recently, rating tags are MS response-enhancing moi- growing attention has been paid to the quan- eties, which are aimed at improving detection titative analysis of endogenous compounds, sensitivity [10] . The exogenous introduction as the levels of endogenous components are of ionizable groups functions as an effec- closely related to the diagnose, treatment and tive way to advance ionization efficiency regulation of cellular functional disorders or in MS, especially for the measurements of human diseases [1] . Current applicable meth- endogenous compounds in trace amount. ods for quantitative analysis of endogenous Moreover, desired LC retention can also be compounds include immunoassays and achieved by integrating hydrophilic or hydro- CE, GC or LC coupled with various detec- phobic tags, which could lead to decreased 19 tion techniques [2,3]. LC–MS has become a and improved chromatographic retention popular analysis technique because of its for strongly and poorly retained compounds, high sensitivity and specificity compared respectively [11] . Matrix effects resulted from with other analytical platforms [4,5]. How- co-elution with other components can be 2015 ever, the establishment of LC–MS methods avoided by incorporating hydrophobic units for the analysis of endogenous compounds to increase the molecular weight and improve can be quite challenging due to the follow- the LC retention [12–14]. For chemically labile ing properties of the analytes: poorly ion- endogenous compounds such as thiols, after ized during MS detection, lack of adequate proper derivatization, better stability in the chromatographic retention, chemical insta- processes of sample preparation and LC–MS bility, presented at low levels and susceptible detection could be achieved [15] . Additionally, to be interfered by matrix components [6,7]. chemical derivatization prior to MS detection To address these issues, chemical derivatiza- could be employed to realize the simultane- tion is introduced to improve the perfor- ous determination of multiple endogenous mance of LC–MS [8]. The process of chemi- compounds. Strategies adopted include the cal derivatization is to modify the original alteration of positive or negative ionization part of

Key terms Amino acids & neurotransmitters Amino acids Ionization efficiency: Rate and degree of the analyte to Amino acids are a class of organic compounds that con- be ionized in ion source interface of the mass spectrometer. tain both amino and carboxy groups. Being the basic SRM: MS/MS provides SRM by selecting both the units of multiple biological macromolecules, they serve precursor ion in the first mass spectrometer stage and as building blocks for proteins, play important roles in the characteristic fragment ion in the second mass spectrometer stage, which significantly enhances the the chemical reactions vital to life and are related to analysis specificity and sensitivity for the analyte in low numerous diseases. Consequently, amino acids analy- abundance. sis has been vested with great significance for metabolomics or proteomics research [23,24], and metabolism properties, and the modulation of the chromatographic disorder investigation [25]. Due to the constant pursuit retention of multiple components. Diverse analytes for more sensitive analysis, LC–MS technique has been exhibit similar chromatographic and mass spectro- widely adopted to assay amino acids, and the reversed- metric properties after derivatization, thus making phase column is the most widely used separation col- possible the simultaneous determination of multiple umn in LC analysis. However, most of amino acids are components under the same LC–MS conditions [16,17]. highly polar analytes, which results in poor retention on Over the past decades, numerous derivatization reversed-phase columns. Analysis without derivatiza- reagents have been reported to facilitate the quantitation by means of classical reversed-phase chromatogra- tive analysis of endogenous compounds. The selec- phy in combination with MS for amino acids is chal- tion of derivatization reagents should be based on the lenging [26]. Moreover, the molecular masses of most objective of the assay [12] . As the labeling efficiency amino acids are in the same range as unspecific sig- greatly depends on critical reaction conditions such nals derived from eluents or matrix, which potentially as temperature, concentration, pH and time, the most leads to undesirable matrix effects [27]. In addition, low demanding step before the submission to MS detection molecular amino acids generally lack diagnostic frag- should be the optimization of a panel of derivatization ment ions for sensitive SRM [28]. As a result, direct conditions [18]. Although the application of chemical determination of amino acids has to face the problems derivatization in bioanalysis is time-consuming, it has of poor LC retention, poor MS ionization and severe been acknowledged as an indispensable technique to matrix effect. Although analysis of underivatized extend the application of LC–MS analysis to a wider amino acids were feasible, classical ion-exchange LC range of analytes [11,19], and increasing emphasis has for polar amino acids required an analysis time of 2–3 been put on the development of derivatization meth- h per sample [26], and high concentrations of the ion- ods for LC–MS analysis to achieve high sensitivity, pair reagent may cause ion suppression [28]. As such, specificity and throughput [20–22]. Considering the much more demanding sample cleanup procedures physiological significance of various endogenous com- were required to eliminate the salts and other compo- pounds, importance should be attached to the accurate nents related to ion suppression [29], and it was likely measurements of their levels in biofluids. The present that the methods without derivatization were not sen- review focuses on the LC–MS combined with chemi- sitive enough to meet specific analytical requirements. cal derivatization methods established for the analysis In order to relieve these analytical puzzles and increase of endogenous compounds in 2010–2015, and a total S/Ns, various derivatization strategies to realize high of six categories of endogenous compounds are dis- m/z ratios for amino acids were established (Figure 1). cussed. We attempted to list and compare the typi- By combination of derivatization, the throughput of cal derivatization reagents and strategies developed LC–MS analysis of multiple amino acids has been successfully to improve the analytical performance significantly improved [30]. for diverse endogenous compounds in recent years, and provided application examples highlighting the Derivatization reagents impact of these important techniques for endogenous For LC–MS analysis of amino acids, the recently compound bioanalysis. reported derivatization reagents included 9-fluore-

Figure 1. Derivatization reactions for amino acids (see facing page). Blue indiatces derivatization targeting amino groups; red indicates derivatization targeting both amino and carboxy groups. DBCEC: 2-(2-[7H-dibenzo (a,g)carbazol-7-yl]-ethoxy)ethyl chloroformate; DBEMM: Dibenzyl ethoxymethylene malonate; DNS-Cl: Dansyl chloride; EBEMM: Benzyl ethyl ethoxymethylene malonate; Fmoc- Cl: 9-fluorenylmethoxycarbonyl chloride; FOSF: 2,5-dioxopyrrolidin-1-yl N-tri(pyrrolidino)phosphoranylideneamino carbamate; TAHS: p-N,N,N-trimethylammonioanilyl N’-hydroxysuccinimidyl carbamate iodide.

2558 Bioanalysis (2015) 7(19) future science group Derivatization methods for LC–MS analysis of endogenous compounds Review

Cl COOH O HN O R O O

Fmoc-CI

O EtO OEt HOOC O OEt NH OEt O DEEMM R OEt

SO2Cl O

H N COOH O2S R N DNS-CI O N

NO HOOC NH NH O O R NH O

N+

I- + TAHS N COOH I-

H2N O R Amino acids NO HOOC NH N NH O O HN+ P N R NH N N O HN+ P N FOSF N

O O O O

O O R1 O O R1 O HN

DBEMM R1 = phenyl R COOH EBEMM R1 = CH3

R COOH O O P NH OH O O P diisopropyl H-phosphite O

O O N Cl O O H O N N COOH O R DBCEC

O O R O O Cl O N H Ethyl chloroformate O O Br O HN 1-bromobutane R

future science group www.future-science.com 2559 Review Zhu, Deng & Zhong

Key term age of C–O bonds upon the introduced derivatization unit [35]; N-phosphorylation labeling amino acids gen- Detection limit: Lowest quantity of the analyte to be erated more intense daughter ions in positive ion mode distinguished from the matrix with confidence S/N. than in negative ion mode, which successively resulted

nylmethoxycarbonyl chloride (Fmoc-Cl) [27,28,31], from the elimination of propylene molecules, H2O and p-N,N,N-trimethylammonioanilyl N’-hydroxysuc- formic acid from the derivatization tag [34]; Fmoc-Cl cinimidyl carbamate iodide (TAHS) [31], 2,5-dioxo- derivatives under negative ion mode displayed two pyrrolidin-1-yl N-tri(pyrrolidino)phosphoranyli- typical fragment ions. One was from the derivatized deneamino carbamate (FOSF) [31], dansyl chloride complex, containing the structure of the amino acid (DNS-Cl) [27,31,32], diethyl ethoxymethylenemalonate and a CO unit from the derivatized tag, and the other - (DEEMM) [27,31], dibenzyl ethoxymethylene malo- product ion corresponded to the [M - H] ion of the nate (DBEMM) [33], benzyl ethyl ethoxymethylene amino acid [28]. malonate (EBEMM) [33], diisopropyl H-phosphite [34], 2-[2-(7H-dibenzo [a,g]carbazol-7-yl)-ethoxy]ethyl Comparison of the derivatization methods chloroformate (DBCEC) [35], 1-bromobutane [36], For LC–MS analysis of amino acids, ‘classical’ isobutyl chloroformate [37], 2,4-dinitrofluoroben- derivatization reagents, for example, DNS-Cl, Fmoc- zene [38] and ethyl chloroformate [39]. All of the above- Cl and DEEMM, were initially developed as the mentioned reagents target amino groups, except ethyl derivatization reagents for UV and fluorescence detec- chloroformate and 1-bromobutane, which readily react tion. Although these reagents are commercially acces- with both amino and carboxy groups. Reagents target- sible and also compatible with MS detection, most of ing carboxy units are useful for the determination of them failed to provide very sensitive analysis results amino group-modified amino acids [36]. Furthermore, (~1 nmol) [31] . Therefore, recent years witnessed a all of the derivatization reagents contain hydrophobic growing trend to develop derivatization reagents that units such as alkyl chains, ester or phenyl groups, ren- are specially designed for mass spectrometric appli- dering amino acids extra hydrophobicity for retention cations since more sensitive analysis is demanded. and separation on reversed-phase columns. Reagents such as TAHS and FOSF have consequently Another challenge posed by the current analytical been reported. By introducing the permanent charges, science is how to precisely quantitate d -amino acids. lower LODs were achieved (~80 fmol) [27,31], and the Chiral derivatization agents have been used for the excess reagent need not be removed since it does not enantiomers separation of chiral amino acids, includ- interfere with the chromatographic analysis and MS ing (S)-N-(4-nitrophenoxycarbonyl)-L-phenylalanine detection. But it must be mentioned that materials 2-methoxyethyl ester [40], R(−)-4-(3-isothiocyana- for TAHS and FOSF synthesis are expensive, and the topyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)- signal of Arg for FOSF was not stable and should not 2,1,3-benzoxadiazole (R(−)-DBD-PyNCS) [41], be considered. More recently, two new derivatization (S)-1-(4-dimethylaminophenylcarbonyl)-3-ami- reagents, DBEMM and EBEMM, have been synthe- nopyrrolidine [42], l -pyroglutamic acid [43] and sized for better ionization and decreased susceptibil- 6-methoxyquinoline-4-carboxylic acid-succinimide ity to matrix effects such as ionization suppression. It ester [44]. is worth mentioning that the LODs after DBEMM derivatization could reach 0.7–19 fmol, lower than Mass spectrometric fragments of the derivatives the detection limits of TAHS and FOSF deriva- For underivatized amino acids, SRM transitions mostly tives, which demonstrated more sensitive quantifica-

rely on the fragments derived from the loss of CO2, tion of amino acids. The derivatization reaction with

H2O, ammonia or formic acid [45,46], which definitely DBEMM proceeds faster in isopropanol than in ace- contributes to the lack of specificity for these methods. tonitrile due to the problem of solubility, and aque- After proper derivatization, diagnostic fragment ions ous 1,1,1,3,3,3-hexafluoro-2-propanol was preferred derived from the reagents or the derivatized complexes to borate buffer owing to the less probability in signal are selected for SRM: in the cases of DNS-Cl, TAHS suppression [33]. and FOSF, derivatives exhibited the identical neutral In the cases of diisopropyl H-phosphite and DBCEC, loss of amino acids moiety, forming three reagent-spe- LC–MS methods coupled with chemical derivatiza- cific ions m/z( 252 for DNS-Cl, 177 for TAHS and 298 tion were developed and employed to screen a total of for FOSF) [31]; in regard to DBEMM and EBEMM, 20 natural amino acids in human serum. The capabil- fragmentation proceeded via loss of neutral benzyl ity of the methods for trace-amount amino acids quan- alcohol from the reagent part [33]; DBCEC derivatives tification was considerably expanded via advantageous showed three intense fragment ions due to the cleav- derivatization, which contributed to the success of

2560 Bioanalysis (2015) 7(19) future science group Derivatization methods for LC–MS analysis of endogenous compounds Review

simultaneous determination of 20 amino acids in com- single run. Cai et al. [16] presented an LC–MS method plex matrix. Diisopropyl H-phosphite based method for analyzing DNSated neurotransmitters and their was applied to examine amino acids related diseases, metabolites. In addition to the enhanced sensitivity since levels of amino acids were probably related to the resulting from the precolumn derivatization, the most disparities in health statuses and metabolism condi- successful attribute of this method was the realization tions. The derivatization reaction was easily performed of simultaneous determination of both monoamines in one-pot reaction under mild conditions within and their acidic metabolites under acidic mobile phase 30 min, and N-phosphorylation labeling was proven in positive ion mode by incorporating electrophilic highly selective for the N-terminal primary amino groups with basic nitrogen atoms. Phenolic hydroxy groups of amino acids and e-amino group of Lys [34]. groups and amino groups of neurotransmitters and More preferably, the complete derivatization reaction of their metabolites are the targets to introduce the DBCEC with amino acids only took 6.3 min at room DNS responsive moieties in the presence of alkales- temperature. This derivatization was applied to inves- cent buffer, but not with an alcoholic hydroxy group. tigate the index of serum amino acids for liver diseases. The established protocol was finally carried out for The results suggested that the levels of eight amino simultaneous determination of ten neurotransmitters acids in hepatitis patients were significantly different and their metabolites in plasma of both schizophrenic from those of healthy subjects, which underlined the patients and healthy subjects. The disparity in plasma determination of these amino acids as prognostic index concentrations of dopamine, and two acidic metabo- for liver diseases [35]. Although derivatization facilitated lites, γ-aminobutyric acid and vanilmandelic acid, simultaneous determination of multiple amino acids, it was revealed, which suggested the importance of should be noticed that identical fragmentations could monitoring their levels when conducting antipsychotic be observed for isobaric or isomeric amino acids as treatment and highlighted the applicability of the the product ions were formed from the derivatization derivatization method for simultaneous determination. tag, which interfered the accurate detection. For these Moreover, recent years witnessed the growing tendency molecules, such as Gln and Lys (m/z 147), Leu and Ile in neurobiology to combine online microdialysis sam- (m/z 132) and Val and Nva (m/z 118), LC separation pling and derivatization strategies prior to LC–MS would be the key to the successful analysis. In addi- analysis. The establishment of online microdialysis tion, Sakaguchi et al. [36] reported 1-bromobutane as of monoamines and amino acids neurotransmitters a derivatization reagent targeting multiple functional using derivatization reagent DNS-Cl [32,48], benzoyl groups (amino, carboxy and phenolic hydroxy groups). chloride [49], propionic anhydride [50], (5-N-succin- 1-Bromobutane derivatization of polar groups effec- imidoxy-5-oxopentyl)triphenylphosphonium bromide tively increased the hydrophobicity of the molecules, (SPTPP) [51] and N-α-Boc-L-tryptophan hydroxy- meanwhile improving the overall basicity of the amino succinimide ester [52], has respectively been reported. acids. The derivatized amino acids, including amino Among these reagents, derivatization with SPTPP pro- group-modified amino acids, could be detected with vided an extremely high sensitivity, which allowed the high sensitivity using LC–MS due to the improved detection of the analyte at the concentration of 1 pM; ionization efficiency (LODs of 5.4–91 fmol). benzoyl chloride appeared as a nonselective derivatization reagent to react with primary and secondary Neurotransmitters amines, phenols and ribose-hydroxy groups, and Neurotransmitters, as the name implies, are chemical allowed nearly all small organic molecule neurotrans- substances acting as messengers in the CNS. Neu- mitters to be labeled. The derivatization methods for rotransmitters mediate a variety of signal transduction amino acids and neurotransmitters are summarized in in CNS to perform the central control. Along with the Supplementary Table 1. fast-developing domain of neurobiology, a large number of neuroactive neurotransmitters have been discovered, Steroids chemically classified into amino acids, monoamines, Steroid hormones peptides and others. The trace-level determination of Steroid hormones play important roles in the pro- neurotransmitters and their acidic metabolites helps cess of body metabolism and immunoregulation [53]. to elucidate the function of signal transduction in Recently, methods based on GC or HPLC with MS CNS and provides information for pathologic diagno- detectors have been generally used for quantitative sis [16,32,47]. For this purpose, LC–MS methods with analysis of endogenous steroid hormones [54]. LC–MS derivatization are applied to improve the detection lim- analysis is considered more applicable than GC–MS, its, and make possible the simultaneous determination for the high temperature of GC may lead to the deg- of neurotransmitters and their acidic metabolites in a radation of some labile steroids. Steroid hormones

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Key term ent, pyridinium moiety and unusual preservation of the pyridine moiety from the derivative while los- Detection-oriented derivatization: Derivatization ing SO . Analyte-dependent product ions for NMPS techniques targeting to improve the detection limits. The 2 most frequently applied strategy is to incorporate MS derivatives contributed to the high specificity for response-enhancing moieties into the analyte. SRM [71] . Girard P and T derivatives bear positive- charged functional groups. Specific SRM for Girard including estrogens, testosterone and progesterone P and T derivatives could be performed using prod- are normally abundant and can be routinely mea- uct ions derived from neutral loss of trimethylamine sured [55]. However, levels of certain type of steroid or pyridine from the dehydrated precursors to increase hormones may occur below current reliable detection the detection sensitivity. However, derivatization with limits among specific populations, for example, peri- or Girard reagent C could not lead to abundant fragment postmenopausal women and geriatric men [56]. Similar ions, and was thus not employed for steroid derivatiza- issues related to accurate steroid measurement compli- tion [69]. Star-Weinstock et al. [70] reported a QAO

cate pediatric diagnosis and therapy for abnormalities reagent containing both aminooxy (-ONH2) group as of pubertal maturation, as the levels of endogenous a carbonyl reactive group and trimethyl ammonium + steroid hormones are often at or below currently avail- ion (-N Me3) as an MS response enhancing group. able detection limits in these individuals [57]. In these The fragment ions selected for SRM of QAO deriva- cases, concentrations of steroids in biological samples tives comprised part of the steroid molecule and part usually reach pM or fM levels [58], and direct LC–MS of the QAO reagent, which imparted more selectivity analysis may not meet the sensitivity requirements as for SRM than the neutral loss fragment (trimethyl- most steroid hormones are neutral compounds and amine), while maintaining the high sensitivity. The may not be efficiently ionized due to the lack of basic LOD of QAO-derivatized analyte was 80-fold lower or acidic functional groups [59–62]. To elevate the detec- than that of nonderivatized analyte. In addition, prac- tion limits, multiple effective derivatization strategies tical derivatization with DAPB was introduced to the have been proposed (Figure 2). steroid hormones having a vicinal diol for sensitivity improvement. DAPB-derivatives were highly respon- Derivatization reagents sive in ESI+ mode and afforded diagnostic product ions Reliable assays of low-level steroid hormones in biologi- during MS/MS fragmentation. Characteristic product cal samples can be realized using various derivatization ions were generated by the cleavage of the bronate ester + strategies. One approach to enhance the sensitivity of and designated to [DAPB + H - H2O - CH3] , [DAPB + + LC–MS based analysis of neutral steroids is to intro- + H - H2O] or [DAPB + H - CH3] , which could be duce an ionizable moiety to the molecules, which is selected based on the intensity for the transitions in exemplified by using DNS-Cl [63–65], picolinic acid [65], SRM mode [66]. 1-methylimidazole-2-sulfonyl (MLDS) chloride [65], pyridinium-3-sulfonyl (PS) chloride [65], (3-dimeth- Comparison of the derivatization methods ylaminophenyl)dihydroxyborane (DAPB) [66], 2-bro- Accurate and reliable assays for low-level unconjugated mopyridine-5-boronic acid (BPBA) [21], 2-hydra- hormones are important because even though their zinopyridine [67] and hydroxylamine [68]. Alternative concentrations are extremely low, fluctuation in their approach involves the preparation of pre-ionized (qua- levels are significant indicator of related physiological ternized) derivatives, so that protonation of the deriva- diseases or even cancers [72,73]. During the process of tive is not required. Suppression of ionization in the ESI derivatization method development for steroid hor- source could be minimized via this type of derivatiza- mones, sensitivity enhancement was the major con- tion. The approach has been reported in studies that cern and derivatization methods were compared in utilized Girard reagents P and T [60,69], quaternary this respect. Girard reagents are frequently used for aminooxy (QAO) reagent [70] and N-methyl pyridin- the detection-oriented derivatization of steroid ium-3-sulfonyl (NMPS) derivatization, which was hormones to form precharged derivatives [60,69]. The prepared via the initial formation of a pyridinium sul- combination of Girard derivatization and LC−MS fonate derivative followed by methylation with methyl technique enabled unequivocal detection of major iodide [71] . endogenous hormones at the pg-level, and Girard reagent T offered slightly (less than twofold) bet- Mass spectrometric fragments of the derivatives ter sensitivity than Girard reagent P [69]. Traditional Among several pre-ionized derivatives, NMPS deriva- derivatization reaction with Girard reagents takes a tives exhibited intense [M]+ ion and three prominent long time to complete at room temperature, so the sam- fragment ions, respectively, corresponding to the par- ples were exposed to microwave irradiation to speed

2562 Bioanalysis (2015) 7(19) future science group Derivatization methods for LC–MS analysis of endogenous compounds Review O OH N OH OH H O OH H H H H H Pyridinium- H H H H H H H H H O S O O O O O S S O O O O O O O S O + N N N N N N Cl Cl Cl O 2 O S Cl S O O SO COOH 1-methylimidazole-2-sulfonyl; PS: 1-methylimidazole-2-sulfonyl; O S O N N N + N N N DNS-CI picolinic acid MLDS chlorid e PS chlorid e NMPS chlorid e OH H H Dansyl chloride; MLDS: chloride; Dansyl H estradiol OH OH HO OH HO HO OH Br N N N N NH O O N NH B B + O O O O N N - + N Br N OH 2

(3-dimethylaminophenyl)dihydroxyborane; DNS-Cl: (3-dimethylaminophenyl)dihydroxyborane; NH Br + N - N H N Br 2 N O H N NHNH + NO N 2 O B BPBA DAPB H B O N Hydroxylamine H HO QAO reagent 2 Girard P reagent HO HO H 2-hydrazinopyridine OH OH OH OH HO H Quaternary aminooxy. H steroid H 17 α , Testosterone Vicinal diol-containing 2-bromopyridine-5-boronic acid; DAPB: 2-bromopyridine-5-boronic O O 20 β -dihydroxypregnenone 3-sulfonyl; QAO: Figure 2. Derivatization reactions for steroids. reactions for 2. Derivatization Figure BPBA:

future science group www.future-science.com 2563 Review Zhu, Deng & Zhong

up the derivatization process. As excess nonreactive specific detection of trace amount of pregnanetriol in Girard reagents interfered with the MS intensity, SPE human urine, which was often used as an indicator was used to resolve the nonreactive reagent interfer- of 21-hydroxylase-deficient congenital adrenal hyper- ence problem [60]. Compared to Girard reagents, newly plasia. The derivatization methods for steroids are reported QAO reagent acclaimed to provide more desir- summarized in Supplementary Table 2. able sensitivity enhancement. Instead of the fragment derived only from the reagent for Girard derivatiza- Vitamin D compounds

tion, more specific fragment ions derived from both the Vitamin D (VD, VD2 in plants and VD3 in mam- derivatization tag and steroid moiety contributed to mals) compounds refer to a group of fat-soluble sec- the lower background noise, thus providing better sen- osteroids responsible for enhancing intestinal absorp- sitivity [70]. In addition, recently developed precharged tion of calcium, iron, magnesium, phosphate and zinc, derivatization strategy to form NMPS derivatives, was thus playing critical role in maintaining bone health observed to offer a twofold increase in response when and regulating inner immune system, cell differentia- compared with the PS derivatives. Though the reaction, emotional cognition and many other biological tions to form NMPS derivatives involved two steps and processes [75,76]. VD itself is not biologically active and required strict evaporation to complete dryness before it exerts its biological effects via further metabolism, critical methylation, the additional derivatization step which poses the importance of quantifying the active for precharging effectively lowered the background metabolites to evaluate the level and metabolic status

noise and conferred extremely high sensitivity (1–10 of VD. In the liver, VD3 is first metabolized to form

fg on column). The method was applied to the quan- 25-hydroxy-VD3, and then principally converted to its

tification of estrogens in the serum of postmenopausal active metabolite 1α,25-dihydroxy-VD3 by the kid- women and older men [71] . ney [77]. Due to the low abundance of the bioactive DNS derivatization is a common strategy adopted dihydroxy metabolites in plasma (pg/ml), most current for endogenous steroid analysis [63–65]. The reac- analytical methods are not capable to provide similar

tion is simple, fast, efficient and sensitive to reach performance for 1α,25-dihydroxy-VD3 or 24,25-dihy-

pg-level. Apart from classical DNS derivatization, droxy-VD3 as they do for 25-hydroxy-VD3 [78]. Hence, other reagents containing the sulfonyl chloride and applicable and sensitive analytical strategies are proton-affinity group were proposed. PS and MLDS required to fulfill the quantification [79]. products exhibited better sensitivity than DNS deriva- Derivatization combined with LC-MS is mostly pre- tives [65]. Hydroxylamine is another commonly-used ferred due to the advantageous improvement in ana- reagent for steroid analysis. The product of hydroxyl- lytical sensitivity and selectivity. The most frequently amine derivatization as oxime derivative was structur- employed derivatization strategy for VD compounds ally similar to that of QAO derivatization. However, is Diels–Alder reaction with 4-phenyl-1,2,4-triazoline- QAO reagent provided better detection sensitivity by 3,5-dione (PTAD), aimed at improving the detection approximately tenfold due to the permanent positive limit of VD and its metabolites [80–87] (Figure 3). Reac- charge of the reagent [70]. In addition, hydroxylamine tion with PTAD results in two epimers (6S and 6R) derivatization involved time-consuming sample prepa- because the reagent reacts with the s-cis-diene moiety ration. The conventional protein precipitation, liquid- from both the α- and β-sides. In this case, epimers liquid extraction and solid phase extraction all failed eluted as one peak would contribute to the increased to eliminate or reduce the matrix effect resulted from detection sensitivity. After derivatization, the m/z ratios hydroxylamine derivatization. The sample had to be are lifted by nearly 200 Da, contributing to the decrease thoroughly cleaned up prior to LC–MS analysis by in background noise. Characteristically, the native VD double extraction using hydrophilic–lipophilic bal- compounds show very rich product ion spectra. On the anced reverse-phase cartridges and mixed mode cation contrary, PTAD-derivatized VD compounds exhibit exchange cartridges [74], or 2-D separation [68]. For ste- only one intensive product ion derived from the cleav- roid hormones containing vicinal diol groups, a detec- age of the C-6–7 bond of the VD skeleton, which tion-oriented derivatization procedure with DAPB has is beneficial for sensitive SRM analysis. After that, been reported [66]. By introducing an easily-charged 4-(4′-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione tag, the MS response was successfully raised by 20- to (DAPTAD), a PTAD analogue having a dimethylamino 160-fold compared with the intact steroids. However, group as a highly proton-affinitive moiety, was reported DAPB derivatization was not suitable for the analy- as a reagent surpassing PTAD in terms of sensitivity sis of catechol compounds such as 4-hydroxyestrone and specificity in LC–MS assay [88]. Although MS/MS due to the rapid hydrolysis. Nevertheless, DAPB fragmentation of DAPTAD derivatives was identical to derivatization followed by LC–MS/MS enabled the that of PTAD-derivatized VD compounds, the high

2564 Bioanalysis (2015) 7(19) future science group Derivatization methods for LC–MS analysis of endogenous compounds Review

OH OH 6S-epimer O H O N N N N N N O HO HO O 25-hydroxy-VD 3 PTAD

O 6R-epimer H N N N

O HO

Figure 3. Derivatization reaction of 25-hydroxy-VD3 with 4-phenyl-1,2,4-triazoline-3,5-dione. PTAD: 4-Phenyl-1,2,4-triazoline-3,5-dione. proton affinity of the dimethylamino group contributed effective chromophoric groups. Instead, LC–MS meth- to the MS response increase by twofold compared with ods employing SRM transition were highly praised the PTAD derivatives, and 40-fold compared with the for the determination of fatty acids. However, there intact VD compounds, which was extremely advanta- existed a major barrier to the LC–MS analysis, as fatty geous in trace-amount analysis of 25-hydroxy-VD3 and acids always underwent unsatisfactory fragmentation. - 1α,25-dihydroxy-VD3. Recently, Hedman et al. [89] Although they could be analyzed as [M - H] in negative reported a new derivatization process using Amplifex ion mode using ESI or atmosphere pressure chemical diene reagent. The Amplifex method was determined ionization, characteristic and sufficient fragmentation to be more sensitive than PTAD derivatization owing was rarely observed via collision-induced dissociation to the quaternary amine functionality, and provided for both saturated and unsaturated fatty acids [22,93], for a tenfold higher S/N than PTAD derivatives. The and in some cases, insufficient ionization in negative method has been successfully applied in the high- ion mode due to moderate gas-phase acidity of the car- throughput measurement of 1α,25-dihydroxy-VD2 and boxy group might also contribute to limited analytical

1α,25-dihydroxy-VD3 in serum. sensitivity [94]. Moreover, the most prominent product

ions generally resulted from the loss of CO2 or H2O Fatty acids from the carboxylic acid group, which brought about Fatty acids are natural carboxylic acids with aliphatic the drawback of low specificity for reliable quantifica- chains. As the essential constituents for lipid biosyn- tion in complicated matrices. As a result, precolumn thesis, intracellular levels of fatty acids are closely derivatization was adopted to supply better sensitivity bound up with many metabolic syndromes, such as and specificity. However, derivatization is not manda- obesity and diabetes [90,91] . Fatty acids are chemically tory for LC–MS analysis of fatty acids. Approaches to divided into saturated or unsaturated according to avoid derivatization and to improve the fragmentation their aliphatic tails. In view of their significant role in behavior when performing LC–MS analysis of fatty regulating physiological functions, considerable efforts acids involved postcolumn addition of metal cations, has been dedicated in last decades to developing precise like barium [95], or using ‘pseudo-molecular’ multiple and sensitive analytical methods for fatty acids. Early reaction monitoring [93]. Nonetheless, quantitative method development focused on GC analysis, but the performance for fatty acids can certainly be enhanced ineluctable high temperature was not appropriate for by salutary derivatization, to enhance the ionization thermo-labile fatty acids [92]. The chromatographic efficiency or to implement fragmentation characteris- assays at room temperature were subsequently devel- tics for SRM [96]. In recent years, various derivatiza- oped by using HPLC. UV detection was difficult to be tion strategies were employed to facilitate the LC–MS applied to the detection of fatty acids due to the lack of analysis of fatty acids (Figure 4).

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R O N+ O

HO methyl iodide N R O N DMAE O

HO O N R O PC R N O (COCI)2 Cl H2N O N R N PA H N OCH3 N HN N N

OCH3 R N OCH3 N N N DMPP O OCH NH2 3 N+ O R HN + O N R HO AMPP

Fatty acids

O O O O NH N 2 H N N R

DAIH O

R H2N HN N HO HN

NO2

NO2 3NPH

Figure 4. Derivatization reactions for fatty acids. Blue: derivatives detected in positive ion mode; yellow: derivatives detected in negative ion mode). 3NPH: 3-nitrophenylhydrazine; AMPP: N-(4-aminomethylphenyl)pyridinium; DAIH: 2-diphenylacetyl-1,3- indandione-1-hydrazone; DMAE: Dimethylaminoethanol; DMPP: 2,4-dimethoxy-6-piperazin-1-yl pyrimidine; PA: 3-picolylamine; PC: 3-pyridylcarbinol.

Derivatization reagents tility of the analytes when performing LC–MS analy- GC–MS analysis of fatty acids usually involved sis of fatty acids. Instead, derivatization strategy aimed derivatization procedures targeting carboxy groups to at sensitivity or selectivity enhancement was the major produce methyl or ethyl esters since fatty acids must concern. Because ionization in the negative ion mode is be transformed into a more volatile form prior to MS inefficient in comparison with that in the positive ion detection. However, there is no need to add to the vola- mode, multiple charge-reversal chemical derivatiza-

2566 Bioanalysis (2015) 7(19) future science group Derivatization methods for LC–MS analysis of endogenous compounds Review tion procedures to improve the ionization efficiency coupling agent, whereas the analytes underwent for the determination of various fatty acids have been unsatisfactory ionization when derivatization was established using positive ion mode ionization, such omitted. Characteristic fragment ions were produced as the use of quaternization derivatization reagent by the cleavage of the carbonyl and amido bond from N-(4-aminomethylphenyl)pyridinium (AMPP) [22,97], the derivatization tag. The optimized derivatization and other nitrogen-containing derivatization reagents, method allowed sensitive mass spectrometric quanti- including 2,4-dimethoxy-6-piperazin-1-yl pyrimi- fication to provide a detection limit of 10 fmol, and dine (DMPP) [94], dimethylaminoethanol (DMAE), was successfully applied to biological samples. 3-picolylamine (PA) and 3-pyridylcarbinol (PC) [98]. In the negative ion mode, 2-diphenylacetyl-1,3-indan- Comparison of the derivatization methods dione-1-hydrazone (DAIH) and 3-nitrophenylhydra- Fatty acids could be generally divided into two cat- zine (3NPH) was used to provide additional sufficient egories, short chain (≤6 carbons in the aliphatic tail) derivatization of FAs [99,100]. and long chain fatty acids. Short-chain fatty acids are recognized as volatile, hydrophilic and highly Mass spectrometric fragments of the polar compounds. Challenges in LC–MS analysis derivatives of short-chain fatty acids include high polarity and For fatty acids, characteristic product ions that ema- limited stability. When it comes to the long chain nated from the fragmentation of the derivatization fatty acids, the major issue in terms of the LC–MS tags were observed after derivatization. In the posi- analysis is the undesirable ionization efficiency due tive ion mode, DMAE ester derivatives exhibited to their relatively high hydrophobicity. For unsatu- intense [M + H]+, which was 72 Da higher than the rated fatty acids, mild conditions are preferred to molecular weights of the intact fatty acids. MS/MS avoid unwanted oxidation or degradation. As a result, fragmentation afforded the most intense product different derivatization strategies were employed to ion [M + 1 - 45]+ derived from the neutral loss of analyze various fatty acids. In the analysis of long the dimethylamine. Trimethylaminoethyl (TMAE) to very long chain fatty acids, TMAE ester iodides ester derivatives were obtained by further reaction of afforded approximately ten times higher MS response DMAE esters with methyl iodide. TMAE derivatives than the corresponding DMAE ester derivatives, and displayed similar product ion composition to DMAE even 100-times in sensitivity when compared with ester derivatives, with the most prominent ion as [M - underivatized fatty acids analyzed in the negative + 59] by neutral loss of the trimethylamine. After that, ion mode [101] . However, this derivatization involved a new derivatization approach to produce picolyl- harmful reagents, and the products lack good chro- amides of fatty acids has been proposed. Here, the matographic resolutions. Li and Franke [98] compared derivatization reagent 3-picolylamine was attached to PA and PC derivatization with TMAE and DMAE the fatty acid via an amide linkage to provide intense derivatization for the LC–MS analysis of fatty acids. protonated molecules in the positive ion mode due The results suggested that PA derivatives provided the to the readily ionizable pyridine group. The prod- best sensitivity and chromatographic property. The uct ion at m/z 109 was formed via the cleavage of derivatization reaction involved two steps. Free fatty amido bond as protonated 3-picolylamine, which acids were first converted to the acyl chloride inter- was selected for SRM transitions [98]. In addition, mediate by the treatment of oxalyl chloride, followed Liu et al. [97] reported an ideal derivatization reagent, by coupling with the amine to form the derivatives. AMPP, which not only delivered enhanced ioniza- After derivatization, the LOD could reach fmol-level tion efficiency, but also supplied informative and for both saturated and unsaturated long chain fatty salutary product ions under MS/MS fragmentation. acids. For polyunsaturated fatty acids such as arachi- The AMPP derivatization tag yielded diagnostic donic acid and linoleic acid, mild derivatization pro- fragment ion at m/z 183.0917 (benzenemethanimine cedure with the charge-reversal reagent, AMPP, lifted pyridinium ion) and 169.0886 (tolylpyridinium dis- the ionization efficiency by ten- to 30-fold compared tonic radical ion), which were useful in identification with the straightforward analysis of intact fatty acids. of the targeted analytes. In the negative ion mode, The LOQs could reach 0.05–6.0 pg [97] or 50–100 fg selective and sensitive SRM for fatty acid detection on-column [22]. Up to 25 fatty acids in mouse serum was achieved by performing precolumn derivatiza- were analyzed using AMPP derivatization, which dis- tion. Neuber et al. [99] reported that the target fatty played its advantage in high-throughput analysis of acids were readily ionized within the ESI source to multiple fatty acids in complex biological samples. give [M - H]- after DAIH derivatization with 1-ethyl- For the analysis of short chain fatty acids, 3NPH was 3-(3-(dimethylamino)propyl)carbodiimide as the employed as a precolumn derivatization reagent to

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convert ten C2–C6 fatty acids to their 3-nitrophenyl- philic interaction chromatography have been proposed hydrazones without the need of reaction quenching for the analysis of these compounds [108,109]. However, before LC–MS analysis. Excellent in-solution stabil- co-elution with endogenous interferents may lead to ity and improved LC retention were achieved for the severe ionization suppression. Therefore, derivatization derivatives. It was observed that 3NPH offered 3- to reagents for tricarboxylic acid cycle intermediates all 25-fold higher detection sensitivities than 2NPH due comprised phenyl group to enhance LC retention. In to higher ESI efficiency [100] . The method was suc- addition to the separation issues, the detection of tri-

cessfully applied to determine ten C2–C6 fatty acids carboxylic acid cycle intermediate in negative ion ESI, in the intestinal tract and fecal samples for the inves- without chemical derivatization, is often problematic. tigation of the complex interplay between diets. The Hence, when analyzed with reversed-phase HPLC derivatization methods for fatty acids are summarized and triple quadruple mass spectrometer, derivatization in Supplementary Table 3. methods were established to enhance the detection sensitivity and separation. Derivatization reagents for LC– Tricarboxylic acid cycle intermediates MS analysis of tricarboxylic acid cycle intermediates Tricarboxylic acid cycle is ubiquitous metabolic process included N-methyl-2-phenylethanamine (MPEA) [107], in living cells of aerobionts. The cycle not only func- O-benzylhydroxylamine (O-BHA) [110], benzyl alcohol tions as the main conjunction for endogenous glycoly- [111], phenylhydrazine [112], O-phenylenediamine [113] sis, lipid and amino acid metabolism, but also plays an and 3NPH [114] . important role in unraveling their metabolic fates and body energy source [102] . Tricarboxylic acid cycle inter- Mass spectrometric fragments of the mediates mainly involve oxaloacetic acid, citric acid, derivatives isocitric acid, oxalosuccinic acid, α-ketoglutarate, suc- Tricarboxylic acid cycle intermediates derivatives dis- cinic acid and fumaric acid. Analytical methods con- played various mass spectrometric patterns. O-BHA cerning various tricarboxylic acid cycle intermediates could be used to derivatize both carboxylic acid and include GC, LC and capillary electrophoresis coupled carbonyl groups, each exhibiting mass gain of 105 Da with UV or MS detectors [103,104]. The most promi- in positive ion mode. The total mass gain depended nent one among these methods is GC–MS, following on the number of acids and carbonyls of the analyte. the derivatization with N-methyl-N-trimethylsilyl- For example, α-ketoglutarate demonstrated mass gain trifluoroacetamide (MSTFA) to increase the volatil- of 315 Da (three units of 105 Da) as it had two car- ity [104] . However, the time-consuming step of phase boxylic acid and one carbonyl groups. After O-BHA transfer involving sample evaporation to dryness and derivatization, MS/MS fragmentation of various deriv- reconstitution is necessary when performing MSTFA atives afforded the intense product ion at m/z 91 (ben- derivatization because this reaction must be conducted zyl from O-BHA) and [M - 123]+ due to the loss of O- in nonaqueous condition, whereas most of the tricar- BHA [110] . When it comes to the MPEA derivatization. boxylic acid cycle intermediates are present in cellular MS/MS experiments performed showed characteristic (aqueous) systems. More importantly, the complicated neutral loss of 135 Da from the introduced derivative process of phase transfer brings about the risk of loss moiety for all the analyzed acids in positive ion mode. of analytes [105] . Considering the resolution, thermal As a result, neutral loss scan could be applied to selec- stability and the compatibility with MS, LC turns out tively detect MPEA derivatives only [107] . In addition, to be the method of choice for tricarboxylic acid cycle derivatization with phenylhydrazine and nitro-substi- intermediates bioanalysis. However, when performing tuted phenyl hydrazine showed different mass spectro- LC–MS analysis, those intermediates usually display metric behaviors [112,114]. Hydrazones were detected in poor chromatographic retention and severe matrix negative ion mode, affording the major fragment ions effects [106], and ionization efficiency can be another of aniline ion, the aniline radical and the precursor ion

unnegligible problem [107] . To overcome the afore- after neutral loss of CO2. mentioned problems, analysts resorted to the concept of derivatization, resulting in multiple derivatization Comparison of the derivatization methods methods for LC–MS analysis of tricarboxylic acid Analysis of tricarboxylic acid cycle intermediates by cycle intermediates (Figure 5). GC–MS in combination with chemical derivatization required nonaqueous condition to conduct the Derivatization reagents derivatization reactions. However, derivatization Tricarboxylic acid cycle intermediates are low molecu- reactions for LC–MS could be conducted in aqueous lar weight polar analytes. Methods based on the use solution, which is advantageous in analyzing cellu- of ion-pair reversed-phase chromatography or hydro- lar tricarboxylic acid cycle intermediates. In general,

2568 Bioanalysis (2015) 7(19) future science group Derivatization methods for LC–MS analysis of endogenous compounds Review

H2N HN O N N OH Phenylhydrazine H R

H N O O 2 O R OH N O O N O H O-BHA R α-keto acids

NH2

N OH NH2

O-phenylenediamine N R

O O O O

Benzyl alcohol O O OH

O OH O N O O O O MPEA HO OH N N OH OH

Citric acid

H2N HN O2N NH H H N OH N NO NO O2N N 2 2 N N

HO OH 3NPH OH

Figure 5. Derivatization reactions for α-keto acids and citric acid. 3NPH: 3-nitrophenylhydrazine; O-BHA: O-benzylhydroxylamine; MPEA: N-methyl-2-phenylethanamine. derivatization methods for LC–MS analysis of tri- tion reactions are often preferred. MPEA was finally carboxylic acid cycle intermediates were established selected to offer desired triply labeled derivatives. The to improve chromatographic behaviors and detec- amide derivative products displayed better ESI+ char- tion limits. Kloos et al. [107] described a derivatiza- acteristics with the LOQs ranging from 12 to 1000 tion method based on the usage of MPEA. A series of nM. However, this method cannot be used to analyze reagents were first examined in terms of the homoge- hydroxy carbolic acids such as glycolic and lactic acid neity of derivatives for di- and tricarboxylic acids, as due to the low sensitivity for the corresponding deriva- the formation of mixtures of mono-, di- and trilabeled tives in positive ion ESI-MS. In order to improve the derivatives for individual acids should be avoided. detection sensitivity of hydroxy carbolic acids, a sensi- Anyway, simple, mild and quantitative derivatiza- tive and reliable derivatization method with 3NPH as

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Key term and progress of human diseases. The significant role of thiol compounds in scavenging reactive oxygen species Biomarker: Endogenous compound which is (ROS) and free radicals adds to the importance of con- physiologically important and indicative of the initiation or progression of a certain type of disease or disorder. ducting bioanalysis for these compounds [116] . As to the analytical techniques for detecting thiol compounds, the derivatization reagent was developed [114] . 3NPH methods based on electrochemistry, CE, GC, LC and provided the best detection sensitivity among three MS have been developed, respectively [117–119]. However, singly nitro-substituted phenylhydrazines (2NPH, there still exist some challenges in analyzing endogenous 3NPH and 4-NPH). Ten central carbon metabolism thiol compounds. Much of the problem lies in the labil- related carboxylic compounds, including glycolate and ity of thiols. The high reactivity of sulphydryl groups is lactate, were derivatized with 3NPH and quantified as the reason for incident conversion of thiols to oxidized biomarkers for metabolomic research, which helped thiols or disulfides via autoxidation. Hence, it is impor- to unveil the potential mechanisms for cellular dys- tant to stabilize these sulphydryl groups in biological function. As hydrazones could significantly amplify samples during the early stages of sample preparation. MS response in the negative ion mode, the favorable Derivatization is a good choice to stabilize the free LOQs (expressed as the on-column account) for vari- thiols and prevent the rapid conversion into oxidized ous analytes reached 0.02–0.2 pmol. For the applica- forms. Recently, derivatization followed by LC–MS tion of 3NPH derivatization, it must be mentioned has been widely utilized for biothiol analysis. How- that cis(E)- and trans(Z)-stereoisomers were observed ever, derivatization was not strictly necessary for thiol for the derivatives of pyruvate and oxaloacetate. The compounds analysis, and few studies have attempted to doublet peaks from a single analyte could possibly analyze thiols by immediately adding EDTA, trichloro- cause analytical errors. Therefore, stable isotope (D acetic acid or stable-isotope internal standard, followed 13 or C) labeled standard compounds used as internal by the storage at -70°C [120,121] . Omitting the derivatiza- standards were required to compensate for the poten- tion simplified the sample preparation, but it could lead tial analytical errors resulting from the existing cis(E)- to excessive autooxidation and unexpected erroneous and trans(Z)-stereoisomers. results and cannot reach high sensitivity. To achieve the It must be pointed out that α-keto acids are unsta- accurate assessment and simultaneous determination of ble and prone to degrade during sample preparation multiple thol components, most LC–MS based analyti- and intracellular concentration determination. In fact, cal methods established for thiol compounds involved accurate measurement of the intracellular concentra- derivatization procedures. In addition, endogenous tions of diverse α-keto acids is difficult. To improve thiol compounds often emerge at low concentrations, the stability of the analytes and facilitate the detection, which definitely increases the difficulty of analysis [122] . derivatization reagents targeting the carbonyl groups Although most of the endogenous thiols naturally con- were used, such as phenylhydrazine and O-BHA, tain carboxy and amino groups, and theoretically could respectively leading to the formation of stable hydra- be ionized by ESI–MS in negative or positive ion mode, zone and oxime. For phenylhydrazine, the derivatiza- the ionization efficiency is relatively poor. In this regard, tion time of 1 h at -20°C was generally sufficient [112]; detection-oriented derivatization was necessary to satisfy for O-BHA, the reaction was conducted for 1 h at the sensitivity requirements. Consequently, various opti- room temperature [110] . The O-BHA derivatives were mized LC–MS methods with derivatization procedures proved to be stable at -20°C for 2 weeks or at room were successively developed to chemically stabilize the temperature for 2 days. Recently, derivatization with sulphydryl groups and amplify the detection sensitivity O-phenylenediamine to convert α-keto acids into of the trace-amount of thiols (Figure 6). stable quinoxaline derivatives has been reported [113] . The stability of quinoxaline derivatives was verified Derivatization reagents at 22°C for 24 h. The derivatization methods for tri- Glutathione and cysteine are two primary thiol com- carboxylic acid cycle intermediates are summarized in pounds, participating in numerous important physi- Supplementary Table 4. ological processes. Due to the lack of endogenous chro- mophore and fluorophore groups, the dominant and Thiol compounds most widely used analytical method for glutathione and Endogenous thiol compounds including glutathione, cysteine is LC–MS. A selective derivatization strategy is cysteine, homocysteine, N-acetylcysteine and cysteinyl- key for the stabilization of the free thiol group and for glycine, fall into the category of bioactive low-molecular- volatile thiol labeling, thus improving detection selectiv- weight substances with sulphydryl groups [115] . So far, ity. Derivatization reagents for glutathione or cysteine most of them are found to be related to the emergence determination included 4-(hydroxymercuri)benzoate

2570 Bioanalysis (2015) 7(19) future science group Derivatization methods for LC–MS analysis of endogenous compounds Review

OH HO Hg O R S Hg PHMB OH

O2N

Cl CF3

O2N

R S CF3 CNBF

O2N O

R N Ph S HSe Dithiothreitol Se Se H RSH recovery N H RSH+ Ph thiols N Ebselen Ph O O

O I O OH S R OH Iodoacetic acid

O Br N – + O Br S N R + – BQB Br

Figure 6. Derivatization reactions for thiol compounds. BQB: ω-bromoacetonylquinolinium bromide; PHMB: 4-hydroxymercuribenzoate.

(PHMB) [123], 4-chloro-3,5-dinitrobenzotrifluoride mode for the precursor and product ions. More impor- (CNBF) [124], methyl or ethyl propiolate [125–127], Se–N tantly, the characteristic and intense fragment ions at containing reagents [15,128] iodoacetic acid [129] and iso- m/z 355 and 311 were confirmed to result from the com- topic pair of reagents (ω-bromoacetonylquinolinium plexes between PHMB and a sulfur atom. The preserva- bromide [BQB] and d7-ω-bromoacetonylquinolinium tion of the Hg–S bond in the product ions definitely 7 bromide [d -BQB]) [130] . Among these reagents, CNBF, reinforced the specificity and confidence of the thiol BQB and iodoacetic acid all have halide leaving group compounds detection, which demonstrated the attrac- that can be easily replaced by the thiol group, leading tive strength of the method [123] . Similarly, derivatiza- to the generation of the stable thioethers; Se–N con- tion with iodoacetic acid also showed fragment ions with taining reagents, such as ebselen, derivatized thiols by the preservation of the derivative moiety by the cleavage breaking the Se–N bond and forming a new Se–S bond. of the original amido bond from the intact thiol com- The formation of Se–S bond was proved to be reversible pounds [129] . The mass spectrometric feature of ebselen with the addition of dithiothreitol. The reversibility of derivatization was the characteristic selenium isotopic the reaction facilitated the isolation of thiol compounds pattern, which was useful in identification and quanti- from complex matrices [128] . fication. Improved ionization could also be achieved by derivatization with ebselen because its aminic nitrogen Mass spectrometric fragments of the significantly contributed to the enhanced MS response. derivatives The diagnostic fragment ion at m/z 275.9922, which Thiol-PHMB complexes were detected in negative ion was identical for various thiol derivatives, was formed 7 mode to provide more desirable S/Ns than positive ion by the cleavage of the Se–S bond [15] . BQB and d -BQB

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are designed as a pair of permanently charged reagents quot were derivatized with BQB, while total thiols in to elevate the signal intensities of the thiol compounds. the other aliquot after a first-step reduction were deriva- The characteristic product ions were formed from the tized with d7-BQB. The two aliquots of derivatization BQB-thiol derivatives by loss of quinoline moiety [130] . products were then mixed and submitted to LC–MS analysis. Derivatization with isotopic reagents offered Comparison of the derivatization methods an efficient strategy to achieve simultaneous determi- Thiols are commonly derivatized before analysis to pre- nation of reduced and oxidized thiols. The sensitivity vent the sulfhydryl autooxidation, improve the LC sep- of the detection was observed to remarkably increase aration, or enhance the MS detection sensitivity. It is by 10- to 40-fold compared with unlabeled thiols in worth mentioning that nM- or fM-level quantification the positive ion mode. The new method was applied for thiol compounds was achieved by employing vari- for the determination of several physiologically impor- ous derivatization methods with LC–MS technique, tant thiol compounds in both reduced and oxidized while the analytical methods without derivatization forms in human urine, and served as an effective tool normally stayed at μM-level. More important was the to quantify the changes in thiol redox. The derivatiza- lability of the sulfhydryl group. Since the issue of sta- tion methods for thiol compounds are summarized in bility was of great importance to ensure the accuracy Supplementary Table 5. and reliability of the bioanalysis of thiol compounds, derivatization methods were compared in terms of the Oxidative stress related biomarkers stability of derivatives. Among the above-mentioned Oxidative stress triggered by ROS is intimately asso- reagents, iodoacetic acid derivatized GSH was observed ciated with a wide spectrum of pathological lesions stable up to 4 days at room temperature [129]; ebselen and diseases [131] . As the initiation of oxidative stress derivatives were proved to be stable at 4–8°C for 4 regulates signaling pathways downstream and alters days [128]; CNBF derivatized thiols were investigated the corresponding physiological indexes, the concept to be stable over 5 days at 4°C [124]; PHMB derivatized of biomarkers has been put forward to evaluate the thiols were proved stable up to 3 months at -20°C [123] . risk [132] . Oxidative stress related biomarkers include In regard to the reaction conditions, derivatization intracellular signaling molecules, lipids and modi- with ebselen was 100% complete within 30 s, which fied biological macromolecules such as DNA damage is a considerable advantage for high-throughput analy- products [133,134]. To some extent, absurd increase or sis [128]; PHMB derivatization was normally com- decrease in the expression levels of various biomark- plete less than 90 s, and large excess of PHMB could ers is indicative of elevated risks of diseases. Therefore, be used without causing interferences [123]; reaction analysis of oxidative stress related biomarkers is of great with CNBF took more than 20 min at room tem- importance to the identification and understanding of perature, and more than 15% organic solvent should the progression of oxidative stress. For this purpose, be added to the reaction medium [124]; for iodoacetic analytical methods were established based on fluo- acid derivatization, the reaction was conducted in the rescence-tagging, ELISA, GC–MS and LC–MS [135] . dark for 1 h at room temperature. The derivatiza- Preliminary treatment with derivatization is one of the tion efficiency of iodoacetic acid,N -ethylmaleimide strategies employed prior to LC–MS analysis, aimed at (NEM) and 5,5′-dithiobis-(2-nitrobenzoic acid) (Ell- improving MS sensitivity and stabilizing certain type man’s reagent, DTNB) was compared. Three reagents of oxidative stress related biomarkers. all provided complete derivatization, while some arti- Damage to biological macromolecules such as factual oxidation was observed for iodoacetic acid and DNA and proteins is the main category of research NEM. However, only iodoacetic acid derivative offered targets as oxidative stress related biomarkers [136] . ideal chromatographic peak, whereas the peak of the Apurinic/apyrimidinic (AP) sites are generally NEM adduct split and poor retention of DTNB deriv- acknowledged as DNA damage products. Previous ative was observed, which enabled iodoacetic acid as analytical studies of AP sites based on ELISA showed the preferable reagent [129] . dramatic discrepancies in the steady-state level rang- 6 In addition, a rapid and sensitive LC–MS/MS ing from 0.67 to 30/10 nt. Li et al. [135] reported a method for simultaneous determination of reduced and novel LC–MS/MS method to rigorously quantify the oxidized thiols in urine samples was reported by using AP sites for risk assessment. By performing enzymatic 7 BQB and d -BQB [130] . Normal determinations of thi- digestion and derivatization of the ring-opened aldehy- ols and oxidized thiols were conducted successively in dic form of the deoxyribosyl moiety with pentafluoro- most cases, and were time-consuming and laborious. phenylhydrazine, the sensitivity has been raised by ten- In this method, each sample was divided into two ali- fold compared with the previous methods. For LC–MS quots before derivatization. Reduced thiols in one ali- analysis of the AP site derivatives, although extensive

2572 Bioanalysis (2015) 7(19) future science group Derivatization methods for LC–MS analysis of endogenous compounds Review fragmentation was observed in negative ion mode, the exist both aldehyde and hydroxy groups in 4-hydroxy- complicated fragmentation did not favor mass spectro- alkenals, their ionization efficiency by ESI-MS is not metric detection. Therefore, more characteristic transi- good enough. Carnosine-adducted 4-hydroxyalkenal tion in positive ion mode was used for SRM to pro- species were easily protonated under acidic conditions vide the detection limit of 6.5 fmol, corresponding to due to the incorporated primary amine, and displayed four AP sites/109 nt in 5 μg of DNA. The established more abundant, informative and characteristic frag- method was successfully applied to evaluate the DNA ment ions from carnosine-adducted polar group. The damage induced by methyl methanesulfonate. combined use of a few of characteristic product ions Lipid peroxidation is a process caused by free radi- could be employed to specifically identify and quan- cals [137] . Biomarkers related to lipid peroxidation have tify these facile oxidation metabolites. Characteristi- been extensively studied [138] . Oxidized phospholipids cally, the ratio of carnosine versus 4-hydroxyalkenals are oxidative stress related biomarkers, stemmed from was critical for the reaction, as a high ratio favored oxidative modifications of low-density lipoproteins complete derivatization, whereas a lower ratio favored caused by ROS [131] . MS of oxidized phospholipids often the reduction of ion suppression from the coexistence involves derivatization of reactive carbonyl groups to of carnosine. The amount of carnosine in 500 times facilitate their enrichment, identification and quantifi- of the 4-hydroxyalkenal levels was settled to offer the cation [19] . Given the very low abundance of this type of best results. In addition, 2,4-dinitrophenylhydrazine biomarkers, Haller et al. [139] employed a dual-function (DNPH) has been frequently used to analyze malo- derivatization reagent incorporating both a hydrazide ndialdehyde and 4-hydroxynonenal [144–146]. As we unit for carbonyl bonding of the oxidized phospholipids have mentioned for 3NPH, derivatization with DNPH and a thiol group for targeting on gold nanoparticles. may also result in analytical error because of the E- The nanoparticle-trapped derivatives were subsequently and Z-stereoisomers caused by the C=N double bond. enriched and finally washed for LC–MS/MS detec- However, purified DNPhydrazones demonstrated tion. After the optimization of related parameters, the only the E-isomers. And a method to transform the LOQs of the two analytes were decreased by 16- and C=N double bond of the DNPhydrazone into a C–N 6-fold, respectively. Despite the innovative derivatiza- single bond by reductive amination using 2-picoline tion strategy to facilitate the enrichment and detection borane, has been reported [147] . The applications of the of the oxidized phospholipids in human plasma and DNPH derivatization based approach involved oxida- low-density lipoproteins, it should be noticed that there tive stress related biomarkers profiling in rat hepato- still existed problems related to the recovery of the ana- cytes, human serum and liver tissues. DNS-hydrazine lytes released from the gold nanoparticles, such as read- has also been reported as the derivatization reagent sorption or irreversible aggregation. Therefore, careful for aldehydes and carbonyls to incorporate easily-pro- derivatization procedure optimization was required to tonated dimethylamino group [148] . In sum, aldehydes eliminate the interference. Another hydrazide reagent, in various biological samples have been investigated 7-(diethylamino)coumarin-3-carbohydrazide (CHH), via robust derivatization strategies, mainly aimed at was used as an effective agent to distinguish between improving stability and detection sensitivity. The hydroxylated and carbonylated phospholipid peroxida- results of above mentioned researches greatly expanded tion products since it reacted specifically with aldehydes the knowledge of the role of aldehydes in risk evalu- and ketones. The MS/MS spectrum displayed charater- ation for oxidative stress. The derivatization methods istic CHH-specific neutral loss of 275 and 260 Da. for oxidative stress related biomarkers are summarized Due to the presence of tertiary amine, the ionization in Figure 7 & Supplementary Table 6. of CHH derivatives in the positive ion mode was also significantly enhanced [140,141] . Conclusion Aldehydes such as hexanal, malondialdehyde, hep- Modern LC–MS based methods have nowadays become tanal, nonanal, 4-hydroxyhexenal or 4-hydroxynon- routine for endogenous compounds analysis. However, enal are a class of oxidative stress related biomarkers even with the most pioneering high-resolution instru- involved in the peroxidation of polyunsaturated fatty ment platform, limited sensitivity or selectivity is con- acids [142] . Derivatization step was employed to avoid stantly encountered during the method development. the degradation of some labile aldehydes, thus improv- Undesirable chromatographic retention and strong ing compound stability to ensure the accurate detection. interference from the matrix components can also be Derivatization of 4-hydroxyalkenals with carnosine to detrimental to the quantitative studies. Derivatization form Michael adducts has been reported to improve the is a critical procedure to improve method sensitivity stability and ionization efficiency, and provide unique or selectivity. Current derivatization strategies mainly fragment ions in MS/MS analysis [143]. Although there include detection-oriented, separation-oriented and sta-

future science group www.future-science.com 2573 Review Zhu, Deng & Zhong

H2NHN F OH OH F F F H HO N F O F HO N OH OH F F 2-deoxy-D-ribose Pentafluorophenylhydrazine F

O O H N X O N SH O n O O – O O n O O O HS NH 2 – O P 12 X N O H O O O O P 12 O O + Oxidized phospholipids N + N

O O NH O O P X NH P X O O 2 11 O O O 11 O N O O HO HO O O

O O O N O CHH HN O Carbonylated lipid peroxidation products O N

O NH2

HN O NH2 NH OH OH N O O OH HN R N O N 4-hydroxyalkenals O OH Carnosine R

O2N NO2

NH2 N O N NO H 2 2

N DNPH N

O O H N O Malondialdehyde 2 HN S N O O HN S N O O N DNS-hydrazine

Figure 7. Derivatization reactions for oxidative stress related biomarkers. CHH: 7-(diethylamino)coumarin-3-carbohydrazide; DNPH: 2,4-dinitrophenylhydrazine; DNS: Dansyl.

2574 Bioanalysis (2015) 7(19) future science group Derivatization methods for LC–MS analysis of endogenous compounds Review bility-oriented derivatization. Applications of derivatiza- in the matrix. In that context, maximum possibility in tion to realize simultaneous determination and chiral sensitivity enhancement can be obtained. Operability – separation have also been reported. As to the selection of regents and buffers should be compatible with MS so as derivatization reagents, major concerns lie in the objec- to simplify the post-treatment prior to LC–MS analysis. tive of the assay and the nature of the target analyte. Mild reaction conditions with minimal by-product for- The chemical structure of the analyte may suggest the mation and analyte loss are preferred when developing reactive function group and potential reaction or reagent or evaluating derivatization reactions and reagents. In suitable for analysis. In addition, the selection also addition, the removal of excess reagents should also be depends on convenience, sensitivity, fragmentation pat- considered when selecting derivatization reagent. Com- terns and LC–MS instruments. Overall, a proper choice plicated extraction to reduce matrix effect as hydrox- for the derivatization reagent could only be reached via ylamine derivatization should be avoided [68,74]. Envi- balanced consideration. Although the optimization of ronmentally friendly – green reagents and reactions are chemical derivatization is time-consuming and labor- preferred to avoid the generation of toxic and harmful intensive, it is generally believed as an effective and com- substances. In all, derivatization methods for LC–MS plementary technique to extend the application of LC– analysis are of great importance to the measurements MS analysis to a wider range of endogenous compounds. of endogenous compounds. Much more efforts should And in recent years, numerous examples where chemical be made to facilitate the derivatization procedure and derivatization is required to improve separation, sensi- enhance the specificity, which contributes to the reliable tivity, selectivity and overall performance of LC–MS and sensitive bioanalysis of endogenous compounds. analysis have been reported. This review focuses on the typical derivatization reagents for LC–MS analysis Future perspective of endogenous compounds, discusses the characteristic Chemical derivatization can be used as a tool to mass spectrometric behaviors of various derivatives, and achieve better sensitivity, which enables studies in compares diverse derivatization methods. small animal models, where sampling volume could In spite of the valuable analytical benefits derived sometimes be limited. Another new area of application from chemical derivatization, it must be recognized that for derivatization method is the analysis of tissue sam- there also exist limitations for chemical derivatization, ples using imaging MS. For example, TAHS, which such as byproduct formation, interference caused by was originally developed for amino acid analysis by excess reagents or analyte loss. Therefore, the benefits, LC–ESI–MS/MS to increase detection sensitivity and as well as the deficiencies of the certain derivatization selectivity, was used as a derivatization reagent to visu- reaction should be carefully evaluated before implemen- alize multiple amino acids in liver sections collected tation. Unfavorable derivatization with severe interfer- from human colon cancer xenografts in vivo [149]. The ence on the target analyte or unimportant improve- chemical derivatization methods established for LC– ments in analytical outcomes should be avoided. For MS will be further expanded to imaging MS, and on- general analytical requirements, conventional analytical tissue chemical derivatization will be an important methods are usually sufficient and preferred due to easy tool in visualization of metabolites on intact tissue, handling. Nonetheless, chemical derivatization could which may lead to an understanding of the distribu- be considered when direct analysis confronts any ana- tion of endogenous metabolites within mammalian tis- lytical difficulty, and the design of new derivatization sues, and can provide insight into the mechanism and regents and methods is still required to achieve optimal location of biological and metabolic processes. analytical performance for LC–MS analysis of endogenous compounds. To this end, future development of Supplementary data derivatization methods should focus on the following To view the supplementary data that accompany this paper aspects: specificity – reagents should be selective for the please visit the journal website at: www.future-science.com/ target analytes with no influence on other components doi/full/10.4155/bio.15.183

Executive summary • Chemical derivatization technology can be very useful in the LC–MS analysis of challenging analytes and could be considered when direct analysis confronts any analytical difficulty. • Chemical derivatization has been employed to improve the performance and applicability of LC–MS in bioanalysis of endogenous compounds. • The integration of derivatization with LC–MS could enhance its capabilities in all steps from the aspects of increasing sensitivity, selectivity, stability and modulating chromatographic retention, thus realizing the high accuracy, high reproducibility and high reliability of bioanalysis.

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Financial & competing interests disclosure ment, consultancies, honoraria, stock ownership or options, The authors have no relevant affiliations or financial in- expert testimony, grants or patents received or pending, or volvement with any organization or entity with a financial royalties. interest in or financial conflict with the subject matter or No writing assistance was utilized in the production of this materials discussed in the manuscript. This includes employ- manuscript.

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future science group www.future-science.com 2581 Methodology

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8 Methodology 2015/12/28 Development of an Excel-based laboratory information management system for improving workflow efficiencies in early ADME screening

Bioanalysis Background: There is a clear requirement for enhancing laboratory information Xinyan Lu management during early absorption, distribution, metabolism and excretion (ADME) Drug Metabolism & Pharmacokinetics, screening. The application of a commercial laboratory information management Platform Technology & Science, GlaxoSmithKline R&D, Shanghai 201203, system (LIMS) is limited by complexity, insufficient flexibility, high costs and extended China timelines. Results: An improved custom in-house LIMS for ADME screening was Tel.: +86 6159 0747 developed using Excel. All Excel templates were generated through macros and [email protected] formulae, and information flow was streamlined as much as possible. This system has been successfully applied in task generation, process control and data management, with a reduction in both labor time and human error rates. Conclusion: An Excel- based LIMS can provide a simple, flexible and cost/time-saving solution for improving workflow efficiencies in early ADME screening.

First draft submitted: 17 August 2015; Accepted for publication: 26 October 2015; Published online: 10 December 2015

The benefit of early absorption, distribution, leaders in laboratory automation software). metabolism and excretion (ADME) screen- Thermo Scientific (company develops and ing is recognized in the pharmaceutical distributes scientific hardware and software industry [1,2]. Increased emphasis on obtain- solutions) has products (Nautilus LIMS, ing in vitro ADME data on new chemical Watson LIMS and Galileo LIMS [7,9–11]) entities has placed greater demands on effi- designed for increasing ADME workflow ciency of ADME assays [3,4]. Implementation efficiency, throughput and data reliabil- of robotics and smart instrument interfaces ity. There are also many LIMS integration has increased ADME screening efficiency strategies for drug discovery [11,12]. 2 in our laboratory over the past few years. These commercial systems are essentially This has resulted in the need to manage either designed for regulatory purpose or large volumes of data, with a complex inter- as generic modular platform that requires face among different functions and priority extensive customization [13] . When it comes 2016 management, as seen in many other labora- to dynamic early discovery environments, tories [5,6]. To meet this requirement, there modules of commercial LIMS are either is a need to replace the relatively outmoded inflexible or they rely heavily upon the com- systems that are using Excel tables with time mercial vendors to provide the expertise consuming manual input, that is prone to to customize the system to the laboratory errors in transcription and calculation. requirements. Commercial LIMS gener- There are numerous laboratory ally require extensive training, have specific information management systems environmental requirements and are reliant (LIMS) [7] in the market, and the representa- upon IT support from outside the labora- tive products are LabWare LIMS ™ and Lab- tory. In addition, adaptation, implementa- Ware ELN ™ (Electronic Laboratory Note- tion, maintenance, integration and future part of book) [7,8] from LabWare (one of the industry updating of these systems is expensive and

Key terms activities approximately eight-fold through minimizing manual intervention and unnecessary communication. Laboratory information management system: A An Excel-based LIMS can provide a simple, flex- laboratory information management system (LIMS) is a software-based system that offers a set of key features ible and cost/time-saving solution for improving work- that support a modern laboratory’s operations. flow efficiencies in early ADME screening. To our best knowledge, there is no commercial LIMS that achieves ELN: An electronic laboratory notebook is a fully electronic replacement of the paper laboratory notebooks used by all of above items without extensive customization. As for discovery. noncommercial LIMS, although comprehensive systems BALTO: A tool that enables scientists to post assay data to for ADME screening exist (e.g., custom LIMS applied database and share their data through the use of querying in Cyprotex [18]), there are no peer-reviewed publications tools. where a custom noncommercial LIMS has been created.

time consuming, which can be a deterrent for smaller Methods groups with limited budgets or urgent timeline [14,15]. Several preconditions of the work environment were For these reasons, a custom in-house LIMS which is required to be met in order to support development straightforward and adaptable may be a solution to of the in house LIMS for ADME screening: active meet our requirements with minimal risks while being laboratory management to ensure efficiency and data more approachable to the general in vitro scientist and quality (label, instrument calibration, consumable bioanalyst in our ADME laboratory. supply, etc.); compliance (data integrity, safety, etc.); Excel was used as the tool to develop this in-house standardized protocols and consistent rules backed by LIMS as it is easy-to-use, system adaptable (can be standard operating procedures; in collaboration with stored in any electronic work space without special suitably trained scientists. installation) and can be updated by anyone with basic Excel and Visual basic for Applications (VBA) [16] Module & template identification training. In addition, Excel is highly compatible Table 1 lists the modules and component templates with most of our instruments (e.g., LC–MS inter- in this in-house LIMS. Information management face, robotic automation equipment) and databases in a general ADME workflow can be completed by (e.g., BALTO, ELN). three modules: task generation, process control and All Excel templates in this custom in-house LIMS data management. A total of six Excel templates were were automated through VBA macros [16] and Excel identified in the above three modules and each Excel formulae [17] . Information transfer was simplified as template was designed to accomplish one specific goal much as possible through template management when in the process. Bespoke report formats were designed copy and paste was required. This system can meet all based upon end-user requirement specifications. The coordination and data management requirements in output format of each template in the information flow an ADME workflow except for LC–MS/MS sequence was designed to be the same as the input format in the generation, as the fully automation of import file is next template to facilitate seamless data transfer and restricted by versatile requirements in discovery stage. reduce human error. The new LIMS has been successfully applied in task generation, process control and data management, and Macro coding or recording made the entire work flow significantly more efficient VBA macros were used only when the required purpose with lower human error rate, reducing time taken for could not be achieved by other in-build Excel func-

Table 1. Modules and templates in Excel-based LIMS for ADME screening. Module no. Module name Template no. Template name Responsible person M1 Task generation T1 Compound list generation Coordinator T2 Request due diligence check Coordinator M2 Process control T3 Request tracking, work Coordinator assignment and monitoring M3 Data management T4 Bioanalytical summary Bioanalyst T5 Parameter calculation, In vitro scientist graphing and archiving T6 Report generation In vitro scientist

100 Bioanalysis (2016) 8(2) future science group Development of an Excel-based LIMS for ADME screening Methodology tions, considering simplicity and system adaptability. the values in drop down lists in T2 (request due dili- Macros were generated ether by VBA coding [16] in T1 gence check), T3 (request tracking, work assignment (compound list generation) or by ‘record a macro’ [19] and monitoring), T5 (parameter calculation, graphing in T3 (request tracking, work assignment and moni- and archiving) and T6 (report generation), which can toring). ‘Record a macro’ is an initiative function in control the type of data or the values that users enter Excel, where each of the commands in Excel that are into a cell, show an ‘input message’ which offers users manually performed are recorded and can be played guidance or display an ‘error alert’ which notifies users back again in an automated manner. that invalid data have been input into the Excel field in T3. Excel formula Excel formula [17,20] was the major tool that used to Conditional formatting develop the in-house system. It is simple, versatile and The versatile Excel function ‘conditional format- easy to understand, so can be a surrogate of macros or ting’ [23] can be used in numerous ways, such as visually pivot tables [21] in many scenarios. Formula can com- explore and analyze data, detect critical issues, identify plete mathematic or logic calculation etc. (Table 2), patterns and trends and can make the template easy- which rely on referencing the contents of other cells. to-read. There are many applications of conditional The cells containing formula are locked to prevent formatting in templates, for example, highlighting user from accident or deliberately deleting or editing contents such as status alert in T3 (request tracking, the formula. work assignment and monitoring), showing duplicates Table 2 lists the name and functions of all formulae results in T2 (request due diligence check), hiding used throughout the templates [17,20]. invalid or zero content in T2, T3, T4 (bioanalytical To identify and correct any logical errors during the summary), T5 (parameter calculation, graphing and development phase, a core group of laboratory per- archiving), underlining unusual values in T5 or using sonnel tested the links and formulae in the templates, color coding to show the classification in T6 (report using representative datasets and performing 100% generation). manual checks on all outputs. Results Data validation The function of the Excel-based LIMS in ADME The Excel function ‘data validation’ [22] can restrict screening workflow what data can be entered into a cell, which makes The Excel-based LIMS includes all of the information the user input more convenient. Data validation was management activities in the ADME screening pro- applied to cells to restrict data entry where possible, to cess. Figure 1 is the schematic of the ADME workflow

Table 2. List of formulae and their function in the Excel templates. Table no. Classification Formula name Purpose and function T2 Statistical Count if Automatically Logic If calculate repeated Show the positions of times repeated items in current Statistical Min, max worksheet

Lookup and ref. Row

Array Array T3 Text Text Automatically analysis Math and trip Sumproduct and graph Automatically calculate accurate turnaround time Date and time Networking days Logic If Analysis of personal Statistic Count if effort T5 Math and trip Round, ABS, linest Calculate ADME parameters and graph Lookup and ref. Vlookup Used to consider species automatically in above

Logic If actions T2: Request due diligence check; T3: Request tracking, work assignment and monitoring; T5: Parameter calculation, graphing and archiving.

future science group www.future-science.com 101 Methodology Lu

DMPK responsibilities

MOSAIC

Compounds In vitro Coordinator Bioanalyst management scientist

Distribute Request In vitro Analyze compounds handling experiment summary

Module 1: task generation Excel-based LIMS Compound list generation for ADME screening

Request due diligence check Module 3: data management

Module 2: process control Bioanalytical summary

Request tracking and Parameter calculation, work assignment graphing and archiving

Monitoring Report generation

DMPK Requestor In vitro representative scientist

Present in Review report Processing project team release archiving

Figure 1. Function of Excel-based laboratory information management system in ADME screening work flow. Same front color in ADME screening work flow and Excel-based LIMS: the step in ADME screening workflow can be completed by corresponding templates in Excel-based LIMS. Front color: ‘green’ for request handling (coordinator); ‘red’ for bioanalytical summary (bioanalyst); ‘blue’ for data management (in vitro scientist); ‘black’ for other activities.LIMS: Laboratory information management system.

in the Excel-based LIMS to show how the Excel-based • Bioanalyst: analyzes samples, input the results and LIMS work effectively with the process flow of ADME summarize the data using T4 in Module 3. screening. In this process, coordinator, in vitro scientist and bioanalyst cooperate to complete the ADME • In vitro scientist: uses Module 3 to generate reports screening as follows: and archive. Detailed explanation of each of the tasks performed • Coordinator: uses Module 1 to generate task, using the LIMS are included in the following section. and uses Module 2 to track request to facilitate work assignment, monitor progress to ensure the Module 1: task generation lead time falls in an acceptable range and provide Typically, each ADME assay is performed after com- periodical statistic result to end users as required. bining all of the requests for a specific assay for all of the different projects. Each project request is entered • In vitro scientist: prepares samples for the by the project team into an individual project specific bioanalyst. workbook. Because the information required is spread

102 Bioanalysis (2016) 8(2) future science group Development of an Excel-based LIMS for ADME screening Methodology

ABCD EFGH I JKL 1 Request descriptions Related requests Repeated requests Comments 2 Folder Project Cpd# Species C & D Repeat (N) Row (min) Row (max) Repeat (N) Row (min) Row (max) Comments 91 20150330 Project1 Cpd1 Human Cpd1Human 2258 91 58 91 To failure 92 Project2 Cpd2 Human Cpd2Human 2 86 92 92 92 93 Project3 Cpd3 Dog Cpd3Dog ab93 93 cd93 ef93

a: IF(COUNTIF(C:C,C93)>1,COUNTIF(C:C,C93),"") b: {=MIN(IF(C$3:C$1000=C93,ROW($3:$1000),65536))} c: {=MAX(IF(C$3:C$1000=C93,ROW($3:$1000),0))} d: IF(COUNTIF(E:E,E93)>1,COUNTIF(E:E,E93),"") e: {=MIN(IF(E$3:E$1000=E93,ROW($3:$1000),65536))} f: {=MAX(IF(E$3:E$1000=E93,ROW($3:$1000),0))}

Figure 2. Example of the output for a ‘request due diligence check.’ Related request (highlighted with yellow in column C): Signifies that the compound has been screened previously (with same or different assay conditions). Repeated request (highlighted with red in column E): Is a request that is identical to an exactly same with previous request.Column A: Reposition of original request emails; Column A-D: Can be a copy from T1 (compound list generation); Column E-K: Can be updated automatically; Column F, I: Number of times this has been repeated; Column G, H, J, K: The min and max row number of related or repeated requests in the sheet. a–f: The detailed information of formulae, which can change based on row by pulling mouse down. across multiple individual Excel workbooks, we need related or repeated requests in the sheet, respectively. If to generate a single combined worksheet that contains related request or repeated requests are identified, T1 all of the relevant information from each of the sepa- should be updated as necessary by the coordinator. rate workbooks. T1 (compound list generation) was developed to automate this task. When T1 is used, two Module 2: process control macros are initiated. First Macro runs and multiple T3 (request tracking, work assignment and monitor- request workbooks (saved in the same folder) are coming) is the dashboard where all of the requests, respon- bined into T1 and become multiple sheets in T1, then sible people, stages, sample or information transfer Macro 2 runs, and multiple sheets in T1 are combined status is viewed. T3 is composed of request record- into a final list. ing, work assignment, statistic results regarding assay, There is the possibility that project may raise request project, etc. (Figure 3). which has been requested previously, either in the same The only sheet which requires any user action is or under different experimental conditions. Manual ‘user input’ sheet. Corresponding columns in T1 (com- checking of these requests can be time consuming. T2 pounds list generation) are transferred to column A and (request due diligence check) (Figure 2) was designed C–G in this page. User input is facilitated by dropdown to check all requests automatically ; highlight any lists and automated calculations. The turnaround time duplicated request which can be investigated to decide calculation is based on the number of working days, if they should be conducted or if the project should from the date the compound was received to the date be informed that the duplicate is not allowed. Cor- of report. It is of note that the folder name in column responding columns in T1 are transferred to columns A is the original storage location which corresponds A–D in T2. All of the historical experiments relevant with column A in T2 (request due diligence check), to a specific compound are highlighted within T2. A facilitating cross worksheet searching. ‘related request’ (highlighted in column C) signifies The ‘monitor page’ shows automatically generated that the compound has been screened previously with statistical result based on assay, projects, turnaround the same or different assay conditions, so the bioana- time and work assignment. This automatically updat- lytical method and relevant historic data can be easily ing style is capable of keep user informed of the lat- referenced (e.g., cpd1 and cpd2 in Figure 2). Part of est progress, which helps end users make informed the ‘related request’ is ‘repeated request’ (highlighted decisions in real time. in column E). This is a request that is identical to an exactly same with previous request, which requires a Module 3: data management clear rationale for the need to repeat the experiment There are three templates in the data management step (e.g., cpd1 in Figure 2). Column A, and F–K show the for each of the ADME assays conducted in our labora- location of the original request emails, lists the number tory. T4 (bioanalytical summary) and T6 (report gen- of times this has been repeated and the row number of eration) use simple functions such as reformatting and

future science group www.future-science.com 103 Methodology Lu a O Turnaround (d) 20150218 Report dat e G Assay3 MN Analyst3 20150211 20150302 20150302 20150302 Receive cpds dat e Excel Assay2 Analysi s Analyst2 h Macros & Programming Requestor Requestor1 Requestor2 Requestor3 Requestor4 f EF Assay1 Analyst1 KL Macro

Analyst Analyst1 Analyst2 Analyst3 Analyst3 Automated J Assay3 ELN # ELN 1 ELN 2 ELN 3 ELN 3 Scientist3 Transient sheet 4-work assignment I CD D Assay2 Scientist1 Scientist2 Scientist3 Scientist3 Assay3 Scientist2 Study director g i In vitro experimen t Automated C Assay2 n1 n2 n3 n4 H Assay1 Scientist1 e Num. of cpds (n) B Assay1 AB Cpd# Cpd1... Cpd2... Cpd3... Cpd4... Assay1 Assay2 Assay3 A Summary 20150211 20150302 20150302 (d) 3-Turnaround time 1 2 3 4 5 6 7 8 20 21 FG Program1 Program2 Program3 Program4 Program# Monitor pages E Assay3 Assay3 Descriptions Description1 Description2 Description3 Description3 CD CD Assay2 Assay2 b c d c Species Species1 Species2 Species3 Species3 Assay1 Assay1 CD Assay2 Assay3 Assay3 Assay1 Study typ e AB AB Tota l Total 1-Summary of assa y 2-Summary of Project 1st quarte r 2nd quarter 3rd quarter 4th quarter Programm 1 Programm 2 Programm 3 Double check Double check DONE 20 21 22 23 24 25 22 23 24 25 26 20 21 ON-GOING Status SCHEDULED SCHEDULED AB 20150211 20150302 20150302 Folder 2 3 4 5 1 a : IF(B2="done",NETWORKDAYS(M2,N2,Information!$G$2:$G$29)+SUMPRODUCT((Information!$H$2:$H$29>=M2)*(Information!$H$2:$H$29<=N2)), "" ) ( Information!$G$2:$G$2 9 ) -holiday; Information!$H$2:$H$29-ajdusted working day b : SUMPRODUCT((TEXT('User Input'!$M$2:$M$1000,"mm")="01")*('User Input'!$B$2:$B$1000="DONE")*('User Input'!$C$2:$C$1000=B$20)*U s er Input'!$H$2:$H$1000) +SUMPRODUCT((TEXT('User Input'!$M$2:$M$1000,"mm")="02")*('User Input'!$B$2:$B$1000="DONE")*('User Input'!$C$2:$C$1000=B$20 ) *'User Input'!$H$2:$H$1000 +SUMPRODUCT((TEXT('User Input'!$M$2:$M$1000,"mm")="03")*('User Input'!$B$2:$B$1000="DONE")*('User Input'!$C$2:$C$1000=B$20 ) *'User Input'!$H$2:$H$1000) c : SUMPRODUCT(('User Input'!$B$2:$B$1000="DONE")*('User Input'!$C$2:$C$1000=B$20)*'User Input'!$H$2:$H$1000 d : SUMPRODUCT(('User Input'!$B$2:$B$1000="DONE")*('User Input'!$C$2:$C$1000=B$20)*('User Input'!$F$2:$F$1000=$A21)*'User Input' ! $H$2:$H$1000 e: SUMPRODUCT(('User Input'!$B$2:$B$1000="DONE")*('User Input'!$C$2:$C$1000=B$20)*'User Input'!$O$2:$O$1000)/SUMPRODUCT(('User In put'! $B$2:$B$1000 ="DONE")*('User Input'!$C$2:$C$1000=B$20)*'User Input'!$R$2:$R$1000); ( R column-auxiliary column with "1" in all cells ) f : The fields are same with “user input”, just for extracting one line request to facilitate further statistics g : IF('Transient sheet'!$I2=B$2,'Transient sheet'!$C2,"" ) h : IF('Transient sheet'!$K2=E$2,'Transient sheet'!$C2,"") i : COUNTIF(B$6:B$1000,$A3) User input

104 Bioanalysis (2016) 8(2) future science group Development of an Excel-based LIMS for ADME screening Methodology

Figure 3. Schematic of the template for ‘request tracking, work assignment and monitoring’, which shows the correlation of ‘user input’ sheet and four monitor sheets (see facing page). In ‘User input,’ Column A: Reposition of original request Emails; Column A, C-G can be a copy from T1 (compound list generation); Column B, I, K, L have dropdown list to facilitate user input; Column O: Show turnaround automatically. Other sheets in ‘Monitor pages’ can be updated automatically. a–i: The detailed information of formulae, which can change based on row or column by pulling mouse down or right.

‘conditional formatting’ for rank ordering or hiding The business process from an information perspec- contents, and ‘dropdown list’ for facilitating user input. tive is streamlined in the Excel-based LIMS, ensur- T5 (parameter calculation, graphing and archiving) ing the output of the previous template can be used can accomplish multiple functions and each ADME directly by pasting into the next template without any assay has at least one T5. To demonstrate the gen- change to the input data order. Therefore, information eral frame work of T5, the template for hepatocytes flow between templates is accelerated and inadvertent metabolic stability assay [24] is shown in Figure 4. ‘Raw errors can be minimized. Information can flow from data’ are the only user input sheet. Copy raw data in T1 (compounds list generation) to T2 (request due dili- T4 to ‘raw data’ sheet in T5 and add additional basic gence check); from T4 (bioanalytical summary) to T5 information as needed (scientists, project name, etc.) (parameter calculation, graphing and archiving); from by selecting from ‘data validation’ drop down lists. T5 to T6 (report generation) and from T5 to the BALTO Other sheets (including ‘derived data’, ‘summary table’ system. Unexpected errors during data transcription and ‘template for BALTO’) are updated automatically were minimized and data integrity was ensured, thus based upon the raw data. A parameter list for different reducing the requirements for any peer-review activities. species is provided (not shown), thus the subsequent calculations in ‘derived data’ and parameters input in Evaluation of Excel-based LIMS ‘template for BALTO’ can automatically adapted to Table 3 compares the workflow between the old process consider species, and do not require user intervention. flow using manual spreadsheets and the new process Contents in ‘summary table’ can be copied to T6 flow using the in-house LIMS, assuming one task con- to generate report quickly. The ‘template for BALTO’ taining 30 compounds from 6 requests. Typically, one is designed to meet BALTO system standards, so that task needs to generate one T1 (compound list genera- data can be copy-pasted to the BALTO to complete the tion), and update T2 (request due diligence check) and results publishing. All files in data management steps T3 (request tracking, work assignment and monitor- are typically uploaded into a centralized record reposi- ing). For the data management step, each task needs to tory, so that they can be easily retrieved and viewed. generate one to two T4s (bioanalytical summary) and All anomalous data points during the entire data man- each request needs to generate one to four T5s (param- agement process are tracked and corresponding notes eter calculation and graphing) and one T6 (report or corrections are made. generation), depending upon the number of compounds and other assay-related requirements (e.g. data The automated process under Excel-based LIMS requested using a different species or cell-type). It is critical to define individual tasks within a frame- This in house LIMS was highly successful in making work of operations to develop any automated process. the entire work flow significantly more efficient, reduc- As shown in Figure 5, the responsible person and cor- ing the time taken for information management activi- responding accountabilities are clear in the process. ties in the ADME screening by approximately eight- All steps were designed to occur in a predetermined fold (from previous 6 h to current ∼50 min). Time timeframe, in which several steps have been identified for each task was notably reduced, owing to the use of to occur automatically. Any deviations are immedi- automated Excel tables and streamlined communica- ately shared with related scientists via Email commu- tion. For the coordinator, who previously needed about nication to ensure awareness and possible follow-up 1 h to complete task generation, tracking and work actions. There are a total of six ‘default places’ facili- assignment, now only needs about 10 min to perform tating materials or information transfer including two the same tasks under the Excel-based LIMS; It takes no sample storage (P3, P4) place and four default location time to complete the periodical summary, as the charts paths (P1, P2, P5, P6). P6 provides centralized secure in the monitor pages in T3 (request tracking, work locations for nonregulated study data which creates a assignment and monitoring) can be copied directly to flexible environment for both accessing and review- the report; For bioanalyst, time to complete the bio- ing study data from any global location. All of above analytical summary is also decreased from the previous can minimize unnecessary communications between 30 min to zero effort. For in vitro scientist, all tasks in scientists thus accelerate the process. data management step can be completed within 1 h,

future science group www.future-science.com 105 Methodology Lu h E TISSUE

K (Char 250) COMMENTS (required) (Char 20) Unit ANNIMAL_GENDER viable cell s U 6 DA ij (Numeric 9.5) (Char 40) E EXPT_LNB_RE F ml/min/g liver mi n ml/min/10 d e f g (required) CA (Char 20) SCALING_FCT_MG_PROT_G_ ANNIMAL_STRAIN IJ GROUPS (Char 50) (Required) R & D CHINA Value Automate d In vitro intrinsic clearance CL-int K_1_MIN c c BA (Numeric 8.5) IJ (Char 20) (Required) (required) Mod (Char 20) Lot1 Lot2 Lot3 ST LOT# INVESTIGATOR ANNIMAL_SPECIES (Char 1) K_MOD AA CD GH T1_2_MIN Cpd1 Cpd2 Cpd3 0.5 (Numeric 15.9) In vitro clint (ml/min/g liver) = ke × ml pe r incubation/# cells per incubation × # cells/g liver Compound (Char 25) Parameter 6 TIMEPOINT STUDY_DESIGN (required) 1/ 2 ke t CL-int CL-int ZA (Numeric 20.16) COMD_CONC_UM B T1_2_MOD 120 (Char 1) GH Species Species1 Species1 Species1 (Char 22) (required) 90 AP14781v1 2 3 1 A PROTOCOL_ID No QR TLAST_MIN (Numeric 8.3) 60 (Char 50) (required) 2 3 4 1 In vitro clint (ml/min/g liver) = Ke × ml per incubation/# cells per incubation × # cells/g liver Summary table Figure Time (min) MILLION_CELLS_M L Automated (required) (Char 100) 30 INVITRO_SYSTEM_UNIT PROJECT_ID 0

-2 -4 -6 -8 Ln (% remaining) (% Ln XY EF 3 (Char 22) 3--1 3--2 3--3 3--4 3--5 3--6 0.5 Cpd3 STUDY_NUM (Numeric 20.14) EF Species1 (required) Ln CMPD_PCT_REMAINING_TLAS T (Numeric 10.7) ab l INVITRO_SYSTEM_CONC (Date) 2--1 2--2 2--3 2--4 2--5 2--6 (required) Cpd2 EXPT_DATE Species1 OP 12 CD 1--1 1--2 1--3 1--4 1--5 1--6 Cpd1 (Char 30) % Remaining Automated (required) (Char 30) Species1 SYSTEM_BATCH_ID PREP_LNB_REF (Numeric 9.5) 0 60 90 15 30 120 Time (min) CALING_FCT_G_TISSUE_KG_BWT CD 1--1 1--2 1--3 1--4 1--5 1--6 (Char 20) ISOFORM k Area ratio (Char 100) Lot1 Lot2 Lot3 LOT# MN 0 B 60 90 15 30 GENERIC_CMPD_NAME 120 Nxxx 20150218 Project1 Scientist1 Time (Char 50) (required) VW HEPATOCYTES IN_VITRO_SYSTEM (Numeric 9.5) AB A L (Char 26) (required) Cpd1 Cpd2 Cpd3 LIVER (Char 50) CMPD_NUMBER TISSUE Cpd1 SCALING_FCT_MI_CELL_G_TISSU ES Species1 Compound 3 2 5 6 7 4 1 2 3 4 1 2 2 3 3 4 4 1 1 Experiment date Investigator ELN# Template for BALTO Project # Raw data a : D2/D$2*100; b Ln(D2); c > or <; d ROUND(ABS(LINEST(E2:E7,B2:B7,TRUE,FALSE)),4); e 0.693/I4; f 0.693/I5*1/0.5*1000; g : IF(A4="human",I6/1000*117.5,"")&IF(A4="dog",I6/1000*170,"")&IF(A4="rat",I6/1000*108,"")&IF(A4="mouse",I6/1000*135,""); h ''Derived Data'!H7 &'Derived Data'!I7 i : VLOOKUP(I4,$A$22:$E$25,2,FALSE); j VLOOKUP(I4,$A$22:$ E$25,3,FALSE); k VLOOKUP(I4,$A$22:$E$25,4,FALSE); l VLOOKUP(I4,$A$22:$E$25,5,FALSE) Derived data

106 Bioanalysis (2016) 8(2) future science group Development of an Excel-based LIMS for ADME screening Methodology

Figure 4. Representative template (hepatocytes stability) for ‘parameter calculation, graphing and archiving’ which show the correlation of different sheets (see facing page). In ‘Template for BALTO,’ Column G, H, L, M, P, Q, R, AC: Information which can be generally fixed; Column B, E, N, O, U: Vacant; Column A, C, D, F, AB, AD: Link from raw data page; Column I, S, T, Z, AA: Link from derived data; Column X, Y: Automated if the last time is 60 min, or will need user action; Column J, K, V, W: Automatically updated based on the species in Column I.a-l: The detailed information of formulae, which can change based on row by pulling mouse down. instead of the previous 3.5 h. The communication time tively supported ADME screening for our discovery required among scientists within one ADME process is portfolio. also dramatically decreased from about 1 h to 10 min. This system is expandable, configurable and flexible. A survey of end users from across the organization For T3 (request tracking, work assignment and moni- indicated that this optimized process was well accepted toring), there is no special volume limit beyond the in- by scientists and easily understood by stakeholders. built limits of Excel (1,048,576 rows for Excel 2007 Several elements in this new system were especially onward) [25]. Changing a cell formula does not need appreciated, such as error prevention measures, faster any major reprogramming skills (Figures 2–4), allow- data turnaround, user friendly interface and improved ing the end users the ability to quickly adapt the LIMS organization. to any minor changes in projects, assays or best prac- tices. Modules can be deleted or added to the LIMS Conclusion & future perspective whenever there is a need for a larger change to the sys- The Excel-based LIMS benefited overall laboratory tem, allowing the laboratory to quickly accommodate efficiency, data quality, data integrity and has effec- any new complex requirements.

Coordinator Task generation Process control Coordinator

Compound list generation Request tracking and work assignment Day 1 P1 P1 Request due diligence check Monitoring

In vitro scientist Experimental part Bioanalyst

P1 Get compounds information Take information Day 1 P2 P2 Create folder for bioanalysis Create sequence

Get the compounds plate Take working solution P3 Day 1 P3 Prepare working solution Tune compounds P2

Day 2 P4 Sample preparation and store Take samples, analyze P4

Bioanalyst Data management Data management In vitro scientist

Parameter calculation, graphing P5, P6 P2 Day 4 Bioanalytical summary and archiving

Report generation

Figure 5. The automated ADME screening process (hepatocytes stability) under Excel-based Laboratory Information Management System with predetermined timeframe, responsible scientists and corresponding tasks and default places (P1-P6). P1: Request folder; P2: Bioanalytical folder; P3: Compound storage; P4: Sample storage; P5: Templates folder; P6: Individual project folder.

future science group www.future-science.com 107 Methodology Lu

Table 3. Comparison of work flow before and after optimization (assume 30 compounds from 6 requests). Tasks Before After Actions Time Actions Time Compound list Open all request files and copy 8 min Open T1, run two macros Within 1 min generation the information to the final list template Request due Use Excel ‘search’ function in a 20 min Copy certain columns in T1 to Within 1 min diligence check semimanual tracking sheet, and corresponding column in T2, related search for the compounds one by request and repeated request can be one. If any related compound is automatically highlighted with detailed identified, further check whether position information this is a repeated request Tracking and work Manually type all data into sheet. 30 min Copy specific columns in T1 to 10 min assignment Manually calculate turnaround corresponding column in T3. Choose time the right items from dropdown list. Automatically calculate turnaround time Monitoring Use filter functions to 2 h/ Run macro in T3 for work assignment Within 1 min semimanually calculate when there month summary, others can be automatically is a requirement updated Bioanalytical Copy raw data of each compounds 30 min Automatically summarized in T4 0 min summary to the summary page one by one, four pastes for one compounds Parameter Derived data only support 1 h Copy information from T4 to 20 min calculation, mathematical calculation corresponding column in T5, parameters graphing and graph can be automatically updated Others are highly manual ‘Summary table’ and ‘template for BALTO’ can be updated without user intervention Report generation Reformat of summary 2 h Copy information from ‘summary table’ 20 min in T5 to corresponding column in T6 Edit project, scientists, etc. Choose from dropdown list Manfully highlight with different Automatically highlight with different color to show classification color to show classification Archiving Reformat the parameters 30 min Copy from specific lines in ‘template for Within 1 min BALTO’ in T5 to BALTO system Input information as required Communication At least six Emails and two face to 1 h Typically <2 emails 10 min effort face meetings T1: Compound list generation; T2: Request due diligence check; T3: Request tracking, work assignment and monitoring; T4: Bioanalytical summary; T5: Parameter calculation, graphing and archiving; T6: Report generation.

It is a straightforward and practical process, with all includes applying versatile Excel tools and minimizing the requisite information to replicate the LIMS, such unnecessary communications. This novel and efficient as major formulae, in the Excel templates (Figure 2–4). concept and methodology have enabled scientists to This system can be replicated by any other laboratory, simplify and reduce their workload. For outsourcing without the need for any complex programming skills. work, there are multiple aspects borrowed from current The essential concepts in custom in-house LIMS LIMS, which are used to handle requests and stream- are ‘automation’ and ‘streamline,’ which include Excel line internal and external communication. There are automation, seamless information transfer and stream- additional applications in laboratory management, lined communication. The corresponding methodology training etc., which would help manage workflow.

108 Bioanalysis (2016) 8(2) future science group Development of an Excel-based LIMS for ADME screening Methodology

This in house LIMS was primarily designed for ment with any organization or entity with a financial interest ADME screening, in which our in house LIMS has in or financial conflict with the subject matter or materi- many advantages over commercial LIMS. This sys- als discussed in the manuscript. This includes employment, tem is expected to be adapted to a wider nonregulated consultancies, honoraria, stock ownership or options, ex- scope and contribute to improving future ways of pert testimony, grants or patents received or pending, or working. royalties. No writing assistance was utilized in the production of this Acknowledgements manuscript. The author would like to thank J Sahi for all her support in developing this custom LIMS, as well as H Liu and Y Li for Ethical conduct of research testing the system. The author would also like to thank R Cal- The author states that they have obtained appropriate institu- loway, Y Zhang and H Liu for their suggestions and comments tional review board approval or have followed the principles in scientific writing. outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations in- Financial & competing interests disclosure volving human subjects, informed consent has been obtained The author has no relevant affiliations or financial involve- from the participants involved.

Executive summary Functions of the in house LIMS • Information management in a general ADME workflow can be completed by three modules: task generation, process control and data management. • A total of six Excel templates were identified as required in the above three modules and each Excel template was designed to accomplish a specific goal in the process. • The output format of each template in the information flow was designed to be the same as the input format in the next template to facilitate seamless data transfer and reduce human error. Major tools used in the in house LIMS • Formulae and macros were applied to reduce data entry, automating calculation and graphing. • Data validation and conditional formatting led to templates that were more convenient and readable. The automated process under the in house LIMS • Elements: consistent rules, default place, predetermined timeframe, clear accountabilities. • Key action: integration, minimize unnecessary communication. System evaluation • This in house laboratory information management system made the entire work flow significantly more efficient, reducing the time taken for activities by approximately eight-fold with less human error rate. • Survey of end users was conducted and confirmed this system as being well accepted and recognized.

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