Benchtop Orbitrap LC-MS/MS

Simultaneous, Fast Analysis of Melamine and Analogues in Pharmaceutical Components Using Q Exactive - Benchtop Orbitrap LC-MS/MS Kate Comstock; Tim Stratton; Hongxia (Jessica) Wang; Yingying Huang Thermo Fisher Scientific, San Jose, CA USA C:\Xcalibur\...\PreF_0_25ppm_FSMS_3 4/20/2012 6:49:14 AM Accucore HILC RSLC C18 100x3 3 um A: H2O B: ACN C: 100 mM Ammonium Acatate KJC

RT: 0.00 - 5.20 SM: 5G NL: 1.87E6 Table 1. Matrix Calibration Standard C:\Xcalibur\...\PreF_0_25ppm_FSMS_3 4/20/2012 6:49:14 AM TIC F: FTMS + p ESI d Full FIGURE 5. Calibration Curves of Neat Mix Standards Overview AccucoreQ Exactive HILC RSLC C18 100x3 Method 3 um A: H2O B:Parameters ACN C: 100 mM Ammonium Acatate KJC ms2 [email protected] Preparation 100 (0.05 ppm to 5 ppm, linear fit) Results [50.00-150.00] MS Purpose: Demonstrate a simple workflow for the simultaneous analysis RT:Full0.00 - 5.20 MSSM: 5G / dd-MS² with polarity switching PreF_0_25ppm_FSMS_3NL: 1.87E6 Confident detection and quantification of melamine and its analogues of melamine and analogues in pharmaceutical components using a Q Resolution: full scan 70,000 FWHW; dd-MS² 35,000 FWHW (at m/z 50 TIC F: FTMS + p ESI d Full were achieved using HR/AM full scan and ms/ms in a data-dependent Exactive benchtop Orbitrap mass spectrometer. 200) ms2 [email protected] 100 fashion with polarity switching. HR/AM full scan data at 70,000 AGC target: 1e6 [50.00-150.00] MS resolution (FWHM) were utilized for quantitation, the high quality full Method: A sensitive, simple and robust method employing high Scan Range (Full MS): 123 to 133 amu PreF_0_25ppm_FSMS_3 resolution mass spectrometry coupled with UHPLC and HILIC mode Relative Abundance RT: 2.71 NL: 1.67E6 scan spectra have sub-2 ppm mass accuracy with external calibration 50 SN: 191 13 15 chromatographic separation. m/z= 127.0721-127.0733 F: and fine isotopic pattern of A+2 isotope ion on C and N for positive FIGURE 2. Sample Preparation Flowchart BP: 127.0728 FTMS + p ESI SIM ms 100 identification (see figure 3 a), which ensure accurate detection and [123.00-133.00] MS Results: Simultaneous detection and quantification of melamine and its Guar Albumin Relative Abundance FIGURE 3. Full Scan and MS/MSRT: 2.71 Spectra of 0.25 ppm MelamineNL: 1.67E6 in quantification of ions of interest in the presence of complicated SN: 191 Genesis analogues in Albumin and Guar gum were achieved with a high degree 5 mg/mL Albumin with sub 2 ppm Error. m/z= 127.0721-127.0733 F: matrices. High resolution accurate mass measurement of signature 50 BP: 127.0728 PreF_0_25ppm_FSMS_3FTMS + p ESI SIM ms of confidence. Weigh 100 mg Albumin, Dissolve 100 product ions were utilized for confirmation of melamine and its Weigh 100 mg Guar, add 5mL [123.00-133.00] MS totally in 5mL water, then vortex Genesis analogues (see figure 3 b). Acetonitrile, vortex for 30 sec. A+2 isotope pattern at mix. 50 70K shows separation of PreF_0_25ppm_FSMS_3 Relative Abundance Isocratic LC separation was conducted using an Accucore HILIC Introduction 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.215 3.4 3.6 3.8 134.0 4.2 4.4 4.6 4.8 5.0 C:\Xcalibur\...\PreF_0_25ppm_FSMS_3 N4/20/2012 and 6:49:14 AM C a) Full Scan Accucore HILC RSLC C18 100x3 3 um A: H2O B: ACN C: 100 mM Ammonium Acatate KJC column. Reproducible baseline separations were obtained within 5 Time (min) 128.0758 NL: Potential drug contamination by melamine and its analogues remains a Relative Abundance 100 4.92E4 minutes. Excellent sensitivity and selectivity were obtained by the 15 13 PreF_0_25ppm_FSMS_3#30 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 902.0 2.2 2.4 2.6 2.8 3.0 N3.2 3.4 3.6 C3.8 4.0 4.2 4.4 4.6 4.8 5.0 1 Add 5 mL water to make a final 4 RT: 2.70 AV: 1 T: FTMS + major concern of the FDA. Currently, the FDA requires that the method p ESI SIM ms [123.00-133.00] HR/AM. The signal to noise ratio at 250 ppb level was 27 for cyanuric 80 Time (min) concentration of 10 mg/ml. Add 15 mL ACN, the final PreF_0_25ppm_FSMS_3 #304 RT: 2.70 AV: 1 NL: 1.49E6 128.0697 used should be suitable for detecting melamine contamination in at-risk 70 Experimental 128.0814 acid and 191 for melamine which exceed FDA specification (S/N > 5:1 Sonicate for 10 min., then add concentration is 5mg/mL, then T: FTMS + p ESI SIM ms [123.00-133.00] 60 components down to at least 2.5 parts per million to give a high degree 50 10ml ACN. Vortex mix for 1 min. vortex mix 1 minute. PreF_0_25ppm_FSMS_3 #304 RT: 2.70 AV: 1 NL: 1.49E6 127.0727 for ions of quantification) (see figure 4). 1 T: FTMS + p ESI SIM ms [123.00-133.00] 40 of assurance that they are not contaminated. RelativeAbundance After settling, transfer top C3 H7 N6 30 127.0727 128.0565 The analyses of albumin fortified with melamine and analogues have solution to Eppendorf tubes. 0.2336 ppm 20 C3 H7 N6 10 good linearity with six-point calibration curves over the range from 50 100 128.0529 128.0897 We present a workflow for simultaneous detection, confirmation, and 0 128.0761 NL: 0.2336 ppm 100 2 100 7.20E2 ppb to 5 ppm with excellent linear regression coefficients (r >0.99), quantification of melamine and its analogues: ammeline, ammelide, and 90 C 3 H6 N6 +H: 15 13 C 3 H7 N6 Prepare matrix calibration Theoreticalp (gss, s /p:40) Chrg 1 80 N C (see figure 6). cyanuric acid (shown in Figure 1) in at-risk pharmaceutical components Prepare matrix calibration 80 R: 70000 Res .Pwr . @FWHM standards (table 1) and control, 70 128.0697 1 standards (table 1) and control, Albumin and Guar gum. A Thermo Scientific Q Exactive benchtop vortex mix 30 sec. 80 60 FIGURE 6. Calibration Curves of Albumin Fortified with For the analyses of guar gum fortified with melamine and analogues, Orbitrap™ mass spectrometer coupled with Thermo Scientific Dionex vortex mix 30 sec. 50 60 40 Melamine and Related Compounds from 0.05 ppm to 5 ppm melamine and ammelide have acceptable quadratic fit for six-point Ultimate 3000 RS UHPLC system were employed in this study. 60 30 calibration curves over the range from 50 ppb to 5 ppm with 20 (linear fit) 2 10 regression coefficients (r >0.99), and for cyanuric acid and ammeline, 40 o 40 0 Melamine CynuricAcid o Place vials in -30 C freezer for 128.05 128.06 128.07 128.08 128.09 128.10 Y = -4221.45+558333*X R^2 = 0.9938 W: 1/X five-point quadratic fit calibration curves were obtained over the range Figure 1. Structures of Melamine and Analogue Place vials in -30 C freezer for m/z Y = 1.37689e+006+2.11909e+007*X R^2 = 0.9930 W: 1/X 30 minutes, then centrifuge at 3000000 Relative Abundance from 0.25 to 5 ppm (cyanuric acid) and 0.05 to 2.5 ppm (ammeline), 30 minutes, then centrifuge at Relative Abundance 17000 g for 30 min. 20 17000 g for 30 min. 20 100000000 2500000 both with good regression coefficients (r2>0.99), (see figure 7). NH NH NH OH 2 2 2 128.0758 80000000 126.8988126.8988 127.0356127.0356127.1110127.1110127.1836127.1836127.2464127.2464 128.0260128.0260128.0758 128.1431128.1431 2000000 0 Compared to commonly used triple quadrupole MS methods, this

0 60000000 1500000 Area N N N N N N N N 126.6 126.7 126.8 126.9 127.0 127.1 127.2 127.3 127.4 127.5 127.6 127.7 127.8 127.9 128.0 128.1 128.2 128.3 Area workflow is simple to set up and allows added post-analysis capability. 126.6 126.7 126.8 126.9 127.0 127.1 127.2 127.3 127.4 127.5 127.6 127.7 127.8 127.9 128.0 128.1 128.2 128.3 Transfer 500 µL supernatant, m/z 40000000 1000000 m/z It avoids the upfront selection of specific compound masses required H N N NH HO N NH HO N OH HO N OH Transfer the supernatant to 2-mL lyophilize to dryness, 20000000 500000 2 2 2 NH2 for SRM methods. This method provides simultaneous detection and vials for injection. reconstitute to 500 µL using PreF_0_25ppm_FSMS_3 #308 RT: 2.72 AV: 1 NL: 1.43E6 0 0 HN N NH quantification of melamine, ammeline and ammelide at 50 ppb level, ACN:H2O 3:1, vortex mix. PreF_0_25ppm_FSMS_3F: FTMS + p ESI d Full ms2#308 [email protected]: 2.72 AV: 1 [50.00-150.00]NL: 1.43E6 2 0 1 2 3 4 5 0 1 2 3 4 5 Ammeline Ammelide Cyanuric Acid 2 Melamine Place in -30 oC freezer for 30 b) dd-MS ppm ppm and 250 ppb level for cyanuric acid, which is well below the FDA C H N O C H N O F: FTMS + p ESI d Full ms2 [email protected] [50.00-150.00] 85.0513 C H N 3 5 5 1 C3H4N4O2 3 3 3 3 3 6 6 + – min. Centrifuge at 17000 g 85.0513C2 H5 N4 Signature Fragment Ion current requirement (2.5 ppm). (M+H)+ (M+H) (M+H)+ (M-H) 128.0102 for 30 min. C2-1.3342H5 N4 ppm 127.0727 128.0567 129.0407 100 Ammeline Ammelide -1.3342 ppm Y = -749953+2.35655e+007*X R^2 = 0.9929 W: 1/X Y = 2639.06+588956*X R^2 = 0.9919 W: 1/X 100 Conclusion 120000000 3000000 80 Transfer the supernatant to 2-mL 100000000 2500000 80 Simultaneous determination, confirmation, and quantitation of Methods vials for injection. 80000000 2000000 60 melamine and its analogues in at-risk pharmaceutical components

N Area Area 60000000 1500000 Material and Reagents H N N Albumin and Guar gum were achieved. This was accomplished by Liquid Chromatography 60 2 40000000 1000000 40 68.0250 using a complete Thermo Scientific solution, consisting of a high Melamine CAS # 108-78-1, Ammelide CAS# 645-93-2, Ammeline CAS# UHPLC Ultimate™ 3000 RS system consisting of : NH 20000000 500000 2 N N 127.0726 resolution Q Exactive benchtop Orbitrap mass spectrometer, Ultimate 645-92-1, and Cyanuric acid CAS#108-80-5, purchased from DGP-3000RS pump Relative Abundance 40 0 0 20 H2N NH2 68.0250 H2N N NH2 0 1 2 3 4 5 0 1 2 3 4 5 3000RS UHPLC system, and Accucore HILIC column. This UHPLC- ChromaDex Inc. California. WPS-3000RS sampler 60.0564 110.0463 127.0726 ppm ppm HRMS MS/MS method is simple, fast, and robust. The combination of Albumin CAS# 9048-46-8, Sigma-Aldrich, p/n A7906. TCC-3000RS column compartment Relative Abundance 200 HR/AM narrow range full scan and dd-MS2 provides rapid, confident Guar CSA# 9000-30-0, Sigma-Aldrich, p/n G4129. 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 60.0564 identification and quantification. Acetonitrile and Water, Fisher. LC Conditions: m/z 110.0463 FIGURE 7. Calibration Curve of Guar Gum Fortified with Ammonium Acetate, Sigma-Aldrich, p/n 73594-25G-F. Thermo Scientific Accucore HILIC Column 100x3 particle (µ) 2.6 0 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 Melamine and Related Compounds, Quadratic fit * Further experiments will be carried out to investigate the potential for Acetic Acid, Sigma-Aldrich, p/n A6283-1L. Thermo Scientific p/n 17526-103030 FIGURE 4. Extracted Ion Chromatogram of Albumin m/z screening of melamine and its analogues in other at-risk Guard cartridge: Accucore ™ HILIC 10x3.0 mm 2.6 µm Thermo Fortified with Melamine and Related Compounds at 0.25 melamine Cyanuric_Acid Scientific p/n 17526-013005 µg/g level Y = 336228+2.74119e+007*X-1.26044e+006*X^2 R^2 = 0.9992 W: Equal Y = 199740+1.60775e+006*X-124693*X^2 R^2 = 0.9990 W: Equal pharmaceutical components using this workflow. Sample Preparation 5000000 Individual stock solutions of melamine, cyanuric acid, ammeline and Mobile Phase A: Water 100000000 4000000 ammelide were prepared at 1.0 mg/mL. Melamine and cyanuric acid Mobile Phase B: Acetonitrile 80000000 In addition, with appropriate sample preparation in place, this HR/AM Mobile Phase C: 100 mM Ammonium Acetate, adjusted to pH 5 3000000

were dissolved in water. Ammeline and ammelide were dissolved in 2 N 60000000 2 Area Area full scan and dd-MS method can be utilized for trace amount material ammonium hydroxide. Standard mixture of 100ng/mL, 1µg/mL and 10 using Acetic acid 2000000 40000000 µg/mL were used to prepare neat and matrix calibration standards. LC program: Isocratic 90% B 10% C for 5.5 minutes identification, confirmation, and quantitation in other applications. Neat standard calibration Flow rate: 0.5 mL/min. 20000000 1000000 o Neat standard mixtures were prepared by serial dilution of 10 Column Temp: 35 C 0 0 0 1 2 3 4 5 0 1 2 3 4 5 µg/mL standard mixture using 3:1 acetonitrile/water to final Injection Volume: 5 µL ppm ppm concentration of 0.25, 1.25, 2.5, 5, 12.5 and 25 ppb, which are Ammeline Ammelide equivalent to 0.05, 0.25, 0.5, 1.0, 2.5 to 5 µg/g of standard mixture Mass Spectrometry Y = -858359+3.3954e+007*X-3.0487e+006*X^2 R^2 = 0.9986 W: Equal Y = 105497+2.8986e+006*X-185363*X^2 R^2 = 0.9989 W: Equal References 10000000 in 5 mg/mL matrix solution. Q Exactive high resolution benchtop Orbitrap MS 70000000

Extracted matrix calibration standards preparation Ion source conditions: 60000000 8000000 1.US FDA Guidance for industry: Pharmaceutical Components at Risk Extracted matrix calibration standards were prepared at 0.05, 0.25, Ionization mode: alternating positive/negative ESI 50000000 6000000 for Melamine Contamination

Ion source: HESI-II 40000000 Area 0.5, 1.0, 2.5, and 5 ppm level by adding standard mixtures to 5 Area 30000000 4000000 2.US FDA LIB 4421 Melamine and Cyanuric Acid Residues in Infant mg/mL Albumin and Guar solutions as shown in Table 1. Sheath gas flow rate: 45 units N2 20000000 Formula October 2008 Matrix effects and percent recoveries were determined by spiking Auxiliary gas flow rate: 10 units N2 2000000 neat solutions into 5 mg/mL Albumin and Guar solutions. Spray voltage (KV): +3.5 for positive, -3.4 for negative 10000000 o 0 0 The sample preparation flowchart is shown in Figure 2. Capillary temp ( C): 320 0.0 0.5 1.0 1.5 2.0 2.5 0 1 2 3 4 5 ppm ppm S-lens RF level: 50.0 * Melamine and Ammelide have 6 point calibration curves in the ranges of 0.05 All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. o Heater temp ( C): 400 to 5 ppm. Cyanuric acid and Ammeline have 5 point calibration curves in the This information is not intended to encourage use of these products in any manners that might ranges of 0.25 to 5ppm and 0.05 to 2.5 ppm, respectively. infringe the intellectual property rights of others.