Quantification of fenoprofen in human plasma using UHPLC-tandem mass spectrometry for pharmacokinetic study in healthy subjects.

A rapid, simple and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed to quantify fenoprofen, a nonsteroidal anti-inflammatory drug in human plasma for a pharmacokinetic study in healthy subjects. Owing to high levels of protein binding, protein precipitation followed by solid-phase extraction was employed for the extraction of fenoprofen and fenoprofen-d3 (used as internal standard) from 200 µL human plasma. Separation was performed on a BEH C18 (50 × 2.1 mm, 1.7 µm) column using methanol-0.2% acetic acid in water (75:25, v/v) under isocratic elution. Electrospray ionization was operated in the negative mode for sample ionization. Ion transitions used for quantification in the selected reaction monitoring mode were m/z 241/197 and m/z 244/200 for fenoprofen and fenoprofen-d3, respectively. Under the optimized conditions, fenoprofen showed excellent linearity in the concentration range 0.02-20 µg/mL (r2 ≥ 0.9996), adequate sensitivity, favorable accuracy (96.4-103.7%) and precision (percentage coefficient of variation ≤4.3) with negligible matrix effect. The validated method was successfully applied to a pharmacokinetic study of fenoprofen in healthy subjects. The significant features of the method include higher sensitivity, small plasma volume for processing and a short analysis time.

Determination of terbinafine in human plasma using UPLC-MS/MS: Application to a bioequivalence study in healthy subjects.

A high-throughput and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the determination of terbinafine in human plasma. The method employed liquid-liquid extraction of terbinafine and terbinafine-d7 (used as internal standard) from 100 µL human plasma with ethyl acetate-n-hexane (80:20, v/v) solvent mixture. Chromatography was performed on a BEH C18 (50 × 2.1 mm, 1.7 µm) column using acetonitrile-8.0 mm ammonium formate, pH 3.5 (85:15, v/v) under isocratic elution. For quantitative analysis, MS/MS ion transitions were monitored at m/z 292.2/141.1 and m/z 299.1/148.2 for terbinafine and terbinafine-d7, respectively, using electrospray ionization in the positive mode. The method was validated according to regulatory guidance for selectivity, sensitivity, linearity, recovery, matrix effect, stability, dilution reliability and ruggedness with acceptable accuracy and precision. The method shows good linearity over the tested concentration range from 1.00 to 2000 ng/mL (r2 ≥ 0.9984). The intra-batch and inter-batch precision (CV) was 1.8-3.2 and 2.1-4.5%, respectively. The method was successfully applied to a bioequivalence study with 250 mg terbinafine in 32 healthy subjects. The major advantage of this method includes higher sensitivity, small plasma volume for processing and a short analysis time.

Development and validation of a rapid and sensitive UPLC-MS/MS assay for the quantification of tofacitinib in human plasma.

A highly sensitive, selective and rapid ultra-performance liquid chromatography-tandem mass spectrometry method has been developed for the quantification of a Janus kinase (JAK) inhibitor, tofacitinib (TOF). The assay employed liquid-liquid extraction with methyl-tert butyl ether to extract tofacitinib and tofacitinib-13C3 15 N (as internal standard) from human plasma. The samples were analyzed on a UPLC BEH C18 (50 × 2.1 mm, 1.7 µm) column using acetonitrile and 10.0 mm ammonium acetate, pH 4.5 (75:25, v/v) as the mobile phase within 1.4 min. The precursor/product ion transitions were monitored at m/z 313.3/149.2 and 317.4/149.2 for tofacitinib and tofacitinib-13C3 15 N, respectively, in the positive electrospray ionization mode. The calibration curves were linear (r2  ≥ 0.9978) across the concentration range of 0.05-100 ng/mL. The mean extraction recovery of tofacitinib across quality controls was 98.6%. The intra- and inter-batch precision (CV) and accuracy ranged from 2.1-5.1 and 96.2-103.1%, respectively. All validation results complied well with the current guidelines. The method is amenable to high sample throughput and was applied to determine TOF plasma concentration in a pharmacokinetic study with 12 healthy Indian subjects after oral administration of 5 mg tablets.

Ultra-performance liquid chromatography-tandem mass spectrometry assay for determination of plasma nomegestrol acetate and estradiol in healthy postmenopausal women.

A highly sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry method is described for the simultaneous determination of nomegestrol acetate (NOMAC), a highly selective progestogen, and estradiol (E2), a natural estrogen in human plasma. NOMAC was obtained from plasma by solid-phase extraction, while E2 was first separated by liquid-liquid extraction with methyl tert-butyl ether followed by derivatization with dansyl chloride. Deuterated internal standards, NOMAC-d5 and E2-d4 were used for better control of extraction conditions and ionization efficiency. The assay recovery of the analytes was within 90-99%. The analytes were separated on UPLC BEH C18 (50 × 2.1 mm, 1.7 µm) column using a mobile phase comprising of acetonitrile and 3.0 mm ammonium trifluoroacetate in water (80:20, v/v) with a resolution factor (Rs ) of 3.21. The calibration curves were linear from 0.01 to 10.0 ng/mL for NOMAC and from 1.00 to 1000 pg/mL for E2, respectively. The intra- and inter-batch precision was ≤5.8% and the accuracy of quality control samples ranged from 96.7 to 103.4% for both analytes. The practical applicability of the method is demonstrated by analyzing samples from 18 healthy postmenopausal women after oral administration of 2.5 mg nomegestrol acetate and 1.5 mg estradiol film-coated tablets under fasting.

Simultaneous determination of etonogestrel and ethinyl estradiol in human plasma by UPLC-MS/MS and its pharmacokinetic study.

A selective, sensitive and rapid ultra-performance liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of etonogestrel (ENG) and ethinyl estradiol (EE) in human plasma. The analytes and their deuterated internal standards, ENG-d7 and EE-d4, were extracted from plasma samples by solid-phase extraction on HyperSep™ Retain PEP cartridges. The chromatographic analysis was performed on an Acquity UPLC HSS Cyano column, 100 Å (50 × 2.1 mm, 1.8 µm), column using gradient mobile phase, acetonitrile and 2.0 mm ammonium trifluoroacetate at 0-1.7 min (65:35, v/v) and 1.8-2.7 min (95:5, v/v) with 0.250 mL/min flow rate. Analytes and IS protonated precursor → product ion transitions (ENG, m/z 325.2 → 257.2; EE, m/z 530.2 → 171.2; ENG-d7, m/z 332.2 → 263.2; EE-d4, m/z 534.2 → 171.2) were monitored on a Triple Quadrupole Mass spectrometer (TQMS), operating in multiple reaction monitoring and positive ionization mode. The calibration curves were established at 10.00-2500 pg/mL for ENG and 1.500-150.0 pg/mL for EE with a correlation coefficient (r2 ) ≥0.9996 for both. The validated method was successfully applied to support a bioequivalence study of 0.15 mg ENG and EE 0.03 mg tablet formulation, administered in 24 healthy Indian females. Method reliability was assessed by reanalysis of 94 incurred study samples.

Simultaneous analysis of glucocorticosteroid fluticasone propionate and its metabolite fluticasone propionate 17ß-carboxylic acid in human plasma by UPLC-MS/MS at sub pg/mL level.

A highly sensitive and rapid ultra performance liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous determination of fluticasone propionate (FP) and its major metabolite, fluticasone propionate-17beta-carboxylic acid (FP 17ß-CA) in human plasma. The analytes and their deuterated internal standards, FP-d3 and FP 17ß-CA-d3 were extracted from 500µL plasma samples by solid phase extraction on Oasis MAX cartridges. The chromatographic analysis was performed on ACQUITY UPLC BEH C18 (50mm×2.1mm, 1.7µm) column using methanol-acetonitrile (50:50, v/v) and 2.0mM ammonium trifluroacetate (ATFA) (85:15, v/v) as the mobile phase. Following separation of the analytes, protonated precursor→product ion transitions (FP: m/z 501.1→293.2, FP17ß-CA: m/z 453.3→293.2, FP-d3: m/z 504.2→293.2, FP 17ß-CA-d3: m/z 456.3→293.2) were monitored on FP 17ß-CA a triple quadrupole mass spectrometer, operating in multiple reaction monitoring (MRM) and positive ionization mode. The calibration curves were established in the range of 0.5-100pg/mL with a correlation coefficient (r2)≥0.9992 for both the analytes. The intra-batch and inter-batch accuracy and precision varied from 95.5-103.4% and 0.74-5.06% across quality controls for both the analytes. The mean assay recoveries for FP and FP 17ß-CA were 84.2% and 93.5% respectively. The validated method was successfully applied to support a bioequivalence study of 200µg FP, administered using nasal spray formulation in 18 healthy Indian subjects. Reproducibility of the method was assessed by reanalysis of 98 incurred study samples.

SPE-UPLC-MS/MS assay for determination of letrozole in human plasma and its application to bioequivalence study in healthy postmenopausal Indian women.

A rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method is described for determination of letrozole in human plasma. Following solid phase extraction (SPE) of letrozole and letrozole-d4 on Orochem DVB-LP cartridges, chromatography was performed on Acquity UPLC BEH C18 (50 mm×2.1 mm, 1.7 µm) column using methanol-0.1% formic acid in water (85:15, v/v) as the mobile phase. Detection was carried out on a triple quadrupole mass spectrometer with an electrospray source, operated under positive ionization mode. Quantitation of letrozole and letrozole-d4 was done using multiple reaction monitoring (MRM) following the transitions at m/z 286.2→217.0 and m/z 290.2→221.0, respectively. The calibration plots were linear through the concentration range of 0.10-100 ng/mL (r2≥0.9990) using 100 µL human plasma. The extraction recovery of letrozole ranged from 94.3% to 96.2% and the intra-batch and inter-batch precision was ≤5.2%. The method was successfully applied to a bioequivalence study of letrozole after oral administration of 2.5 mg tablet formulation to 16 healthy postmenopausal Indian women. The assay reproducibility was also established through incurred sample reanalysis (ISR) of 74 subject samples.

SPE-UPLC-MS/MS assay for determination of letrozole in human plasma and its application to bioequivalence study in healthy postmenopausal Indian women$ / 药物分析学报

A rapid and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method is described for determination of letrozole in human plasma. Following solid phase ex-traction (SPE) of letrozole and letrozole-d4 on Orochem DVB-LP cartridges, chromatography was per-formed on Acquity UPLC BEH C18 (50 mm ? 2.1 mm, 1.7 mm) column using methanol-0.1%formic acid in water (85:15, v/v) as the mobile phase. Detection was carried out on a triple quadrupole mass spec-trometer with an electrospray source, operated under positive ionization mode. Quantitation of letrozole and letrozole-d4 was done using multiple reaction monitoring (MRM) following the transitions at m/z 286.2-217.0 and m/z 290.2-221.0, respectively. The calibration plots were linear through the con-centration range of 0.10–100 ng/mL (r2Z0.9990) using 100 mL human plasma. The extraction recovery of letrozole ranged from 94.3% to 96.2% and the intra-batch and inter-batch precision was r 5.2%. The method was successfully applied to a bioequivalence study of letrozole after oral administration of 2.5 mg tablet formulation to 16 healthy postmenopausal Indian women. The assay reproducibility was also established through incurred sample reanalysis (ISR) of 74 subject samples.

Repeat analysis and incurred sample reanalysis: recommendation for best practices and harmonization from the global bioanalysis consortium harmonization team.

The A7 harmonization team (A7 HT), a part of the Global Bioanalysis Consortium (GBC), focused on reviewing best practices for repeat analysis and incurred sample reanalysis (ISR) as applied during regulated bioanalysis. With international representation from Europe, Latin America, North America, and the Asia Pacific region, the team first collated common practices and guidance recommendations and assessed their suitability from both a scientific and logistical perspective. Subsequently, team members developed best practice recommendations and refined them through discussions and presentations with industry experts at scientific meetings. This review summarizes the team findings and best practice recommendations. The few topics where no consensus could be reached are also discussed. The A7 HT recommendations, together with those from the other GBC teams, provide the basis for future international harmonization of regulated bioanalytical practices.

Analysis of 21-hydroxy deflazacort in human plasma by UPLC-MS/MS: application to a bioequivalence study in healthy volunteers.

A sensitive and rapid ultra performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method has been developed for the determination of 21-hydroxy deflazacort in human plasma using betamethasone as the internal standard (IS). After solid-phase extraction from 100 µL human plasma, the analyte and IS were analyzed on Waters Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 µm) column using acetonitrile-4.0mM ammonium formate, pH 3.5 (90:10, v/v) as the mobile phase. The protonated analyte was quantified by selected reaction monitoring in the positive ionization mode by triple quadrupole mass spectrometer. The calibration plots were linear over the concentration range 0.50-500 ng/mL. Intra-batch and inter-batch precision (% CV) and accuracy (%) for five quality control samples ranged within 1.40-4.82% and 98.0-102.0% respectively. The overall mean extraction recovery of 21-hydroxy deflazacort from plasma ranged from 95.3 to 97.3%. Matrix effect was assessed by post-column analyte infusion and the extraction recovery was >95.0% across four quality control levels for the analyte and IS. Stability was evaluated under different conditions like bench top, autosampler, processed sample (at room temperature and in cooling chamber), freeze-thaw and long term stability. The method was applied to support a bioequivalence study of 30 mg deflazacort tablet formulation in 28 healthy subjects. Assay reproducibility was demonstrated by reanalysis of 115 incurred samples.

UPLC-MS/MS assay for the simultaneous quantification of carvedilol and its active metabolite 4'-hydroxyphenyl carvedilol in human plasma to support a bioequivalence study in healthy volunteers.

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the simultaneous determination of carvedilol and its pharmacologically active metabolite 4'-hydroxyphenyl carvedilol in human plasma using their deuterated internal standards (IS). Samples were prepared by solid-phase extraction using 100 µL human plasma. Chromatographic separation of analytes was achieved on UPLC C18 (50 × 2.1 mm, 1.7 µm) column using acetonitrile-4.0 mM ammonium formate, pH 3.0 adjusted with 0.1% formic acid (78:22, v/v) as the mobile phase. The multiple reaction monitoring transitions for both the analytes and IS were monitored in the positive electrospray ionization mode. The method was validated over a concentration range of 0.05-50 ng/mL for carvedilol and 0.01-10 ng/mL for 4'-hydroxyphenyl carvedilol. Intra- and inter-batch precision (% CV) and accuracy for the analytes varied from 0.74 to 3.88 and 96.4 to 103.3% respectively. Matrix effect was assessed by post-column analyte infusion and by calculation of precision values (coefficient of variation) in the measurement of the slope of calibration curves from eight plasma batches. The assay recovery was within 94-99% for both the analytes and IS. The method was successfully applied to support a bioequivalence study of 12.5 mg carvedilol tablets in 34 healthy subjects.

Challenges in the simultaneous quantitation of sumatriptan and naproxen in human plasma: application to a bioequivalence study.

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the simultaneous determination of sumatriptan and naproxen in human plasma using naratriptan and indomethacin as the internal standards (ISs). The plasma samples were prepared by solid phase extraction on Phenomenex Strata-X cartridges using 100 µL human plasma sample. Chromatography was carried out on Waters Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 µm) analytical column under isocratic conditions using a mobile phase consisting of methanol-acetonitrile-4.0mM ammonium acetate (70:10:20, v/v/v). The precursor→product ion transition for both the analytes and ISs was monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ionization mode. The method was validated over a wide dynamic concentration range of 0.050-100 ng/mL for sumatriptan and 0.050-100 µg/mL for naproxen. Matrix effect was assessed by post-column analyte infusion and the extraction recovery was >95.0% across four quality control levels for both the analytes. Stability was evaluated under different conditions including bench top, processed sample, freeze and thaw and long term. The method was applied to support a bioequivalence study of 85 mg sumatriptan+500 mg naproxen sodium fixed dose formulation in 28 healthy Indian subjects. Assay reproducibility was demonstrated by reanalysis of 123 incurred samples.

Enantiomeric separation of verapamil and its active metabolite, norverapamil, and simultaneous quantification in human plasma by LC-ESI-MS-MS.

A simple, selective and high-throughput liquid chromatography-tandem mass spectrometry method has been developed and validated for the chromatographic separation and quantification of (R)- and (S)-enantiomers of verapamil and its active metabolite, norverapamil, in human plasma. All four analytes along with deuterated internal standards (D(6)-verapamil and D(6)-norverapamil) were extracted from 50 µL human plasma by liquid-liquid extraction. Separation was achieved on a Chiralcel OD-RH (150 × 4.6 mm, 5 µm) analytical column with resolution factors of 1.4 and 1.9 for (R)- and (S)-enantiomers of verapamil and norverapamil, respectively. A mobile phase consisting of 0.05% trifluoroacetic acid in water-acetonitrile (70:30, v/v) afforded capacity factors of 2.45, 3.05, 2.27 and 3.13 for (R)- and (S)-enantiomers of verapamil and norverapamil, respectively. Detection was carried out on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion modes. The method was validated over the concentration range of 1.0-250.0 ng/mL for all four analytes. Absolute recovery for the analytes ranged from 91.1 to 108.1%. Matrix factors calculated at three quality control levels varied from 0.96-1.07. The method was successfully applied to a pharmacokinetic study in 18 healthy Indian males after oral administration of a 240-mg verapamil tablet formulation under fasting conditions.

Simultaneous quantitation of HIV-protease inhibitors ritonavir, lopinavir and indinavir in human plasma by UPLC-ESI-MS-MS.

A selective, sensitive and high-throughput ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method has been developed and validated for the quantification of HIV-protease inhibitors ritonavir (RTV), lopinavir (LPV) and indinavir (IDV) in human plasma. Sample clean-up involved protein precipitation of both drugs and fluconazole used as internal standard from 100 µL human plasma. All the analytes were chromatographically separated on a Waters Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 µm particle size) analytical column using 0.1% formic acid and methanol (40:60, v/v) as the mobile phase. The parent → product ion transitions for ritonavir (m/z 721.40→ 296.10), lopinavir (m/z 629.40→ 447.40) and indinavir (m/z 614.4→ 421.0) IS (m/z 307.10 → 220.10) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive ion mode. The method was validated over the concentration range of 30-15,000 ng/mL for LPV and IDV and 3-1500 ng/mL for RTV. The method was successfully applied to a pilot bioequivalence study in 36 healthy human subjects after oral administration of lopinavir 200 mg and ritonavir 50 mg tablet formulation under fasting conditions.

Validation of a liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood.

A liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method was validated for the quantification of Sotrastaurin (AEB071) and N-desmethyl-sotrastaurin in human blood. The validation of the analytical procedure was performed according to the latest Food and Drug Administration (FDA) "Guidance for Industry, Bioanalytical Method Validation". Chromatographic separation was performed using an RP C18 (50 mm × 4.6 mm, 5 µm) column at 40±3.0 °C with a mobile phase consisted of 2 mM ammonium acetate in water (pH 4.5):methanol:acetonitrile (25:15:60, v/v) of a flow rate of 1 mL/min followed by quantification with tandem mass spectrometer, operated in electrospray ionization (ESI) positive ion mode and applying multiple reaction monitoring (MRM). The validated method described in this paper presents high absolute recovery, with a sensitivity of 3.00 ng/mL as lower limit of quantitation using a sample volume of 300 µL, low inter-run bias and variability (for Sotrastaurin, -4.4 to 0.4% and 1.8 to 2.5% and for N-desmethyl-sotrastaurin, ranged from 1.6 to 2.3% and 2.7 to 3.9%, respectively) with a short runtime of 3.5 min. The method was validated using K3EDTA as specific anticoagulant and cross-validated using Li-Heparin and Na-Heparin. The method was specific for Sotrastaurin and N-desmethyl-sotrastaurin within the given criteria of acceptance (apparent peak area for Sotrastaurin and N-desmethyl-sotrastaurin in zero samples ≤ 20% of mean peak area at LLOQ) in human blood. The method was fully validated for the quantitative determination of Sotrastaurin and its metabolite N-desmethyl-sotrastaurin in human blood between the range of 3.00 ng/mL and 1200 ng/mL.

Highly sensitive and rapid ultra-performance liquid chromatography-tandem mass spectrometry method for the determination of nifedipine in human plasma and its application to a bioequivalence study.

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed for the determination of nifedipine in human plasma using nifedipine-d6 as the internal standard (IS). The plasma samples were prepared by solid-phase extraction on Phenomenex Strata-X cartridges employing 200 µL human plasma. Chromatography was carried out on Waters Acquity UPLC BEH C18 (50 × 2.1 mm, 1.7 µm particle size) analytical column under isocratic conditions using a mobile phase consisting of 4.0 mm ammonium acetate-acetonitrile (15:85, v/v). The precursor → product ion transitions for nifedipine (m/z 347.2 → 315.2) and IS (m/z 353.1 → 318.1) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring and positive-ion mode. The method was validated over a wide dynamic concentration range of 0.050-150 ng/mL. Matrix effect was assessed by post-column analyte infusion and the mean extraction recovery was 95.6% across four quality control levels. The method is rugged and rapid with a total run time of 1.2 min and was applied to a bioequivalence study of 20 mg nifedipine tablet formulation in 30 healthy Indian subjects under fasting condition. Assay reproducibility was confirmed by reanalysis of 116 incurred samples.

Application of a validated ultra performance liquid chromatography-tandem mass spectrometry method for the quantification of darunavir in human plasma for a bioequivalence study in Indian subjects.

A simple, precise and rapid ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for the quantification of darunavir, a protease inhibitor, using darunavir-d9 as internal standard (IS). The method involved liquid-liquid extraction of darunavir and IS in methyl-tert-butyl ether from 50 µL human plasma. The chromatographic separation was achieved on an Acquity UPLC BEH C18 (50 mm × 2.1mm, 1.7 µm particle size) analytical column under gradient conditions, in a run time of 1.6 min. The precursor → product ion transitions for darunavir (m/z 548.1 → 392.0) and IS (m/z 557.1 → 401.0) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was extensively validated for its selectivity, sensitivity, carryover check, linearity, precision and accuracy, reinjection reproducibility, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. The linearity of the method was established in the concentration range of 1.0-5000 ng/mL. The mean relative recovery for darunavir (100.8%) and IS (89.8%) from spiked plasma samples was consistent and reproducible. The application of this method for routine measurement of plasma darunavir concentration was demonstrated by a bioequivalence study conducted in 40 healthy Indian subjects for a 600 mg tablet formulation along with 100mg ritonavir as booster under fast and fed conditions. To demonstrate the reproducibility in the measurement of study data, an incurred sample reanalysis was done with 400 subject samples and the % change in concentration was within ± 12%.

Development and validation of a sensitive and rapid method to determine naratriptan in human plasma by LC-ESI-MS-MS: application to a bioequivalence study.

A simple, sensitive, selective, and rapid high-performance liquid chromatography-tandem mass spectrometry method is developed and validated for the quantitation of naratriptan, using sumatriptan as internal standard (IS). The method included liquid-liquid extraction of naratriptan and IS with methyl-tert-butyl ether and dichloromethane mixture from 100 µL human plasma. The chromatographic separation is achieved on ACE C18 (50 mm × 2.1 mm, 5 µm) analytical column under isocratic conditions, using 0.1% acetic acid and acetonitrile (15:85, v/v) at a flow-rate of 0.4 mL/min. The precursor → product ion transitions for naratriptan (m/z 336.10 → 98.06) and IS (m/z 296.09 → 251.06) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The linearity of the method for naratriptan is determined in the range of 103-20690 pg/mL with the analysis time of 1.5 min. The method is fully validated according to USFDA guidelines. A systematic post-column infusion study is conducted for ion-suppression due to endogenous matrix components. The process efficiency of analyte (96%) and IS (93%) from spiked plasma samples was consistent and reproducible. The application of the method is demonstrated by a bioequivalence study of 2.5 mg naratriptan tablet formulation in 31 healthy volunteers under fasting conditions.

Validated ultra-performance liquid chromatography tandem mass spectrometry method for the determination of pramipexole in human plasma.

A sensitive and high throughput ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS-MS) method has been developed for the determination of pramipexole, a dopamine agonist, in human plasma. Sample preparation involved liquid-liquid extraction of pramipexole and ranitidine as the internal standard (IS) in ethyl acetate from 100 µL human plasma. The chromatographic separation is achieved on a Waters Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 µm) analytical column using an isocratic mobile phase, consisting of 10 mM ammonium formate (pH 7.50)-acetonitrile (15:85, v/v), at a flow-rate of 0.5 mL/min. The precursor → product ion transition for pramipexole (m/z 212.1 → 153.0) and IS (m/z 315.0 → 176.1) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over a wide dynamic concentration range of 20-4020 pg/mL. Matrix effect is assessed by post-column infusion experiment and the process efficiency were 91.9% and 85.7% for pramipexole and IS, respectively. The method is rugged and rapid with a total run time of 1.5 min and is applied to a bioequivalence study of 0.25 mg PPX tablet formulation in 30 healthy Indian male subjects under fasting condition.