Application Note: 314 Fast Quantitative Analysis of Pergolide Using APCI on the TSQ Quantum Discovery Nicola Hughes,1 Antony Harvey,2 Witold Winnik,3 and Gary Paul3 1Biovail Contract Research, Toronto, Ontario, Canada; 2Thermo Fsher Scientific, San Jose, CA, USA; 3Thermo Fsher Scientific, Somerset, NJ, USA Overview relative to those obtained at unit mass-resolution.4,5,6 The Key Words In this report, the APCI source is used to demonstrate increase in analyte sensitivity achieved using enhanced mass-resolution analysis is also accompanied by an • TSQ Quantum the TSQ Quantum’s ability to perform fast quantitative extended linear dynamic range for these assays.4,6 Discovery™ analysis of another ergoline derivative, pergolide. An ana- lytical method is developed which is suitable for quantita- • APCI Method tive analysis of pergolide in plasma following oral Goals • High-throughput administration at all possible dosage regimens (low to 1. Develop a fast, sensitive bioanalytical method using APCI. Analysis high dose). The results of unit and enhanced mass-resolu- 2. Demonstrate a broad linear dynamic range for the assay. tion analysis on the TSQ Quantum are also compared to 3. Use enhanced mass-resolution capability to lower • Linear Dynamic demonstrate sensitivity improvements that can be made to method LLOQ. Range analytical methods using the enhanced mass-resolution feature of the triple quadrupole mass spectrometer. • Pharmacokinetic Experimental Conditions Application Introduction Chemicals and Reagents: Pergolide mesylate (purity >98%) was supplied by Sigma Chemical Company (St. Louis, MO, Pergolide (Figure 1) is a synthetic ergoline derivative that USA). Cabergoline (purity >99%) was chemically synthe- is used for the treatment of Parkinson’s disease, providing sized. HPLC-grade acetonitrile and methanol, and reagent therapeutic activity at doses as low as 0.75 to 3.0 mg per grade ammonium acetate were purchased from EM Sciences day.2,3 However, due to the severe side effects of pergolide (Gibbstown, NJ, USA). Bovine plasma was acquired from treatment, therapy is often initiated at daily doses of Sigma Chemical Company. only 0.05 mg. The development of simple, fast, accurate, and precise Standard and Sample Preparation: Stock solutions of methods for the determination of potent drugs, such as pergolide and the internal standard cabergoline (Figure 1), pergolide, requires ultimate performance from an analytical were each prepared at concentrations of 1 mg/mL in technique. Additionally, for a method to be of practical methanol and stored at –25°C. A plasma solution was use for analysis following any given dosage, the method prepared by precipitating bovine plasma with a 2× volume must have a broad dynamic range, preferably without of acetonitrile. A 10 µg/mL pergolide plasma standard was detector saturation at higher analyte concentrations. prepared by spiking the precipitated bovine plasma with Although ESI has found widespread application in the the pergolide stock solution. Working plasma standards development of sensitive detection methods for bioana- were then prepared by sequentially diluting the 10 µg/mL lytical applications, it is more prone to matrix suppression pergolide plasma standard with the precipitated bovine than APCI and often requires longer chromatographic run plasma solution to produce a series of standard concen- times to compensate. In this study, the APCI source was used in combination with a relatively high chromato- H graphic flow rate (0.8 mL/min) to assess the performance O N CH3 of the Thermo Scientific TSQ Quantum Discovery in the CH3 development of a sensitive detection method with an O N N CH3 extremely short run time (~1 minute). H CH2SCH3 Additionally, the performance in the enhanced mass- H H resolution mode of the TSQ Quantum Discovery for the N N CH2CH2CH3 CH2 quantitative analysis of pergolide in plasma is tested. Utility H H of enhanced mass-resolution can achieve mass separation of an analyte of interest from isobaricmatrix/chemical inter- HN HN ferences in the H-SRM experiment. This can result in a Pergolide Cabergoline further improvement in quantitative performance for (Internal Standard) analytes present in complex biological matrices, and improved LLOQ (lower limit of quantitation) sensitivities Figure 1: Structures of pergolide and cabergoline trations of greater than five orders of magnitude (50 pg/mL The pergolide SRM conditions were as follows: to 10 µg/mL). Prior to analysis, the pergolide standards Parent Mass: m/z 315 were spiked with cabergoline such that each standard had Product Mass: m/z 208 a fixed internal standard concentration of 100 ng/mL of Scan Width: 0.6 u cabergoline. The plasma standards were then ready for Scan Time: 0.12 s direct injection into an HPLC—no further sample clean Collision Energy: 25 eV up was necessary. Q1 Peak Width (enhanced mass-resolution): Sample Analysis: HPLC analysis was performed on a 0.20 u FWHM Thermo Scientific Surveyor™ LC System. The chromato- Q1 Peak Width (unit mass-resolution): graphic separation was performed using isocratic condi- 0.70 u FWHM tions on a Thermo Scientific BetaBasic™ 18, 5 µm column Q3 Peak Width: 0.70 u FWHM using a mobile phase of methanol/water/formic acid The cabergoline SRM conditions were as follows: (98:2:0.1). The method used an LC flow rate of 0.8 Parent Mass: m/z 452 mL/min, and the injection volume was 5 µL. Product Mass: 381 Detection was performed on the TSQ Quantum Scan Width: 0.6 u Discovery triple quadrupole mass spectrometer equipped Scan Time: 0.12 s with the APCI source. The mass spectrometer was Collision Energy: 19 eV operated under both unit and enhanced mass-resolution Q1 Peak Width (enhanced mass-resolution): conditions, and the APCI settings were optimized to 0.20 u FWHM obtain the highest [M+H]+ abundance. Q1 Peak Width (unit mass-resolution): 0.70 u FWHM Mass Spectrometry Q3 Peak Width: 0.70 u FWHM Instrument: TSQ Quantum Discovery Source: APCI Results and Discussion Ion Polarity: Positive The quantitative results for pergolide in plasma obtained Discharge Current: 17 µA at unit mass-resolution on the TSQ Quantum Discovery APCI Vaporizer Temperature: 420°C are shown in Figures 2–4 and in Table 1. Under unit mass- Sheath/Auxiliary Gas: Nitrogen resolution conditions, the LLOQ for pergolide was 500 fg Sheath Gas Pressure: 65 arbitrary units on column (Figure 2), where no interfering peaks were Auxiliary Gas Pressure: 0 arbitrary units observed in the corresponding extracted ion chromatogram Capillary Temperature: 250°C of the blank plasma (Figure 3). In a previously developed Scan Type: SRM LC/ESI/MS method for the quantitation of pergolide in Collision Gas: Argon plasma, an LLOQ of 5 pg/mL was reported on an older Collision Gas Pressure: 1.3 mTorr generation triple quadrupole mass spectrometer, which 100 90 80 70 Pergolide 60 SRM 315 → 208 50 S/N 26 40 30 20 Relative Abundance 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Time (min) 100 90 80 Cabergoline (ISTD) 70 SRM 452 → 381 60 S/N 689 50 40 30 20 Relative Abundance 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Time (min) Figure 2. LC/APCI/SRM chromatogram of 500 fg on column of pergolide (m/z 315 → 208) and 500 pg on column of cabergoline internal standard (m/z 452 → 381) in plasma under unit mass-resolution conditions Page 2 of 6 represented 3 pg of analyte on column.7 Hence, the on the TSQ Quantum Discovery, minimally-treated running of this assay by APCI on the TSQ Quantum samples were analyzed considerably faster (1 minute run Discovery results in a significant improvement in detection time, Figure 2) using a very small injection volume (5 µL). limit. Previous comparative APCI/SRM studies have shown A linear dynamic range covering five orders of magnitude analyte sensitivities with up to an order of magnitude was achieved with a correlation coefficient of R=0.998, improvement on the TSQ Quantum, relative to the older using a weighting factor of 1/x2 (Figure 4). For many generation TSQ 7000™ at unit mass-resolution.8,9 Such triple quadrupole mass spectrometers, linearity over three improvements in detection limits means that the same orders of magnitude poses no problem for analytes in target LLOQ can be achieved using a much lower per- solution (no matrix), but detector saturation is commonly assay plasma volume and/or a simpler extraction proce- observed with extracted plasma samples, which therefore dure. For example, the previous ESI quantitation method restricts the practical range of the method. The TSQ required a very high plasma volume (1.5 mL), a highly Quantum Discovery, with the ability of providing greater selective extraction/enrichment procedure, and a longer than five orders of linear dynamic range, provides the fol- run time of 3.5 minutes.7 In the current APCI experiment lowing significant advantages: 100 90 80 Plasma Blank 70 SRM 315 → 208 60 50 40 30 20 Relative Abundance 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Time (min) 100 90 80 70 Plasma Blank 60 SRM 452 → 381 50 40 30 20 Relative Abundance 10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Time (min) Figure 3. LC/APCI/SRM chromatograms of drug-free plasma under unit mass-resolution conditions 300 250 200 150 Area Ratio 100 50 0 0 10000 20000 30000 40000 50000 pg on Column Figure 4. Calibration curve for pergolide in plasma under unit mass-resolution conditions covering five orders of linear dynamic range (500 fg to 50 ng on column), R=0.998 using 1/x2 weighted regression Page 3 of 6 • Bioanalytical methods covering > three orders of linear from –5.3% to 11.0% and 0.5% to 5.0%, respectively.