Priyanka A. Shah

LC–tandem mass spectrometry method for the simultaneous determination of metformin and sitagliptin in human plasma after ion-pair solid phase extraction

A reversed-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was established for simultaneous determination of two oral hypoglycemic drugs metformin (MET) and sitagliptin (STG) in human plasma. The analytes were extracted from 50μL human plasma by ion-pair solid phase extraction using sodium lauryl sulphate on Phenomenex Strata-X (30mg/1mL) cartridges. The chromatographic separation was accomplished on XSelect HSS CN (150×4.6mm, 5μm) column using mobile phase consisting of methanol-8.0mM ammonium formate in water, pH 4.5 (80:20, v/v) under isocratic condition. Tandem MS detection was performed on a triple quadrupole spectrometer equipped with an electrospray ionization source, operated in the positive mode. Multiple reaction monitoring (MRM) was used to quantify the analytes following transitions, m/z 130.1→60.1 and m/z 408.3→235.1 for MET and STG respectively. The method displayed acceptable linearity in the concentration range of 4.00-3200ng/mL for MET and 1.00-800ng/mL for STG. The intra-batch and inter-batch precisions were ≤5.1% and accuracy ranged from 96.5 to 103.3% for both the drugs. The mean recovery of MET and STG obtained from spiked plasma samples was 82.5% and 90.4% respectively with minimal matrix interference. Both the drugs were found to be stable under all mandatory storage conditions. The validated method was successfully applied to a clinical pharmacokinetic study for a fixed-dose tablet formulation containing 500mg MET and 50mg STG in 16 healthy volunteers.

Simultaneous quantification of atenolol and chlorthalidone in human plasma by ultra-performance liquid chromatography-tandem mass spectrometry.

A simple, sensitive and reproducible ultra-performance liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous determination of atenolol, a β-adrenergic receptor-blocker and chlorthalidone, a monosulfonamyl diuretic in human plasma, using atenolol-d7 and chlorthalidone-d4 as the internal standards (ISs). Following solid-phase extraction on Phenomenex Strata-X cartridges using 100 μL human plasma sample, the analytes and ISs were separated on an Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 µm) column using a mobile phase consisting of 0.1% formic acid-acetonitrile (25:75, v/v). A tandem mass spectrometer equipped with electrospray ionization was used as a detector in the positive ionization mode for both analytes. The linear concentration range was established as 0.50-500 ng/mL for atenolol and 0.25-150 ng/mL for chlorthalidone. Extraction recoveries were within 95-103% and ion suppression/enhancement, expressed as IS-normalized matrix factors, ranged from 0.95 to 1.06 for both the analytes. Intra-batch and inter-batch precision (CV) and accuracy values were 2.37-5.91 and 96.1-103.2%, respectively. Stability of analytes in plasma was evaluated under different conditions, such as bench-top, freeze-thaw, dry and wet extract and long-term. The developed method was superior to the existing methods for the simultaneous determination of atenolol and chlorthalidone in human plasma with respect to the sensitivity, chromatographic analysis time and plasma volume for processing. Further, it was successfully applied to support a bioequivalence study of 50 mg atenolol + 12.5 mg chlorthalidone in 28 healthy Indian subjects.

Fast and sensitive LC–MS/MS method for the simultaneous determination of lisinopril and hydrochlorothiazide in human plasma

A sensitive and rapid liquid chromatography-tandem mass spectrometry (LC–MS/MS) method has been developed for the simultaneous determination of lisinopril (LIS) and hydrochlorothiazide (HCTZ) in human plasma using their labeled internal standards (ISs). Sample pre-treatment involved solid phase extraction on Waters Oasis HLB cartridges using 100 µL of plasma, followed by liquid chromatography on Hypersil Gold C18 (50 mm×3.0 mm, 5 µm) column. The analytes were eluted within 2.0 min using acetonitrile-5.0 mM ammonium formate, pH 4.5 (85:15, v/v) as the mobile phase. The analytes and ISs were analyzed in the negative ionization mode and quantified using multiple reaction monitoring. The method showed excellent linearity over the concentration range of 0.50–250.0 ng/mL for both the analytes. The intra-batch and inter-batch precision (% CV) was ≤5.26% and their extraction recoveries were in the range of 96.6%–103.1%. Matrix effect evaluated in terms of IS-normalized matrix factors ranged from 0.97 to 1.03 for both the analytes. The validated method was successfully applied to determine the plasma concentration of the drugs using 10 mg lisinopril and 12.5 mg hydrochlorothiazide fixed dose formulation in 18 healthy Indian volunteers.

Determination of silodosin and its active glucuronide metabolite, KMD-3213G in human plasma by LC-MS/MS for a bioequivalence study

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous determination of silodosin (SLD) and its active metabolite silodosin β-d-glucuronide (KMD-3213G) in human plasma. Liquid-liquid extraction of plasma samples was carried out with ethyl acetate and methyl tert-butyl ether solvent mixture using deuterated analogs as internal standards. The extraction recoveries of SLD and KMD-3213G were in the ranges 90.8-93.4 and 87.6-89.9%, respectively. The extracts were analyzed on a Symmetry C18 (50 × 4.6 mm, 5 μm) column under gradient conditions using 10 mm ammonium formate in water and methanol-acetonitrile (40:60, v/v), within 6.0 min. For MS/MS measurements, ionization of the analytes was carried out in the positive ionization mode and the transitions monitored were m/z 496.1 → 261.2 for SLD and m/z 670.2 → 494.1 for KMD-3213G. The method showed good linearity, accuracy, precision and stability in the range 0.10-80.0 ng/mL for SLD and KMD-3213G. The IS-normalized matrix factors obtained were highly consistent, ranging from 0.962 to 1.023 for both analytes. The method was used to support a bioequivalence study of SLD and its metabolite in healthy volunteers after oral administration of 8 mg silodosin capsules.

LC–MS/MS analysis of metformin, saxagliptin and 5‐hydroxy saxagliptin in human plasma and its pharmacokinetic study with a fixed‐dose formulation in healthy Indian subjects

A specific and rapid liquid chromatography-tandem mass spectrometry method is proposed for the simultaneous determination of metformin (MET), saxagliptin (SAXA) and its active metabolite, 5-hydroxy saxagliptin (5-OH SAXA) in human plasma. Sample preparation was accomplished from 50 μL plasma sample by solid-phase extraction using sodium dodecyl sulfate as an ion-pair reagent. Reversed-phase chromatographic resolution of analytes was possible within 3.5 min on ACE 5CN (150 × 4.6 mm, 5 μm) column using acetonitrile and10.0 mm ammonium formate buffer, pH 5.0 (80:20, v/v) as the mobile phase. Triple quadrupole mass spectrometric detection was performed using electrospray ionization in the positive ionization mode. The calibration curves showed good linearity (r2  ≥ 0.9992) over the established concentration range with limit of quantification of 1.50, 0.10 and 0.20 ng/mL for MET, SAXA and 5-OH SAXA respectively. The extraction recoveries obtained from spiked plasma samples were highly consistent for MET (75.12-77.84%), SAXA (85.90-87.84%) and 5-OH SAXA (80.32-82.69%) across quality controls. The validated method was successfully applied to a bioequivalence study with a fixed-dose formulation consisting of 5 mg SAXA and 500 mg MET in 18 healthy subjects. The reproducibility of the assay was demonstrated by reanalysis of 87 incurred samples.

An improved LC–MS/MS method for the simultaneous determination of pyrazinamide, pyrazinoic acid and 5-hydroxy pyrazinoic acid in human plasma for a pharmacokinetic study

In the present work the plasma levels of PZA and its two active metabolites, pyrazinoic acid (PA) and 5-hydroxy pyrazinoic acid (5-OH PA) were determined by a sensitive and rapid LC-MS/MS method. The analytes and their labeled internal standards were extracted from 200μL plasma samples by liquid-liquid extraction with methyl tert-butyl ether: diethyl ether (90:10, v/v) under acidic conditions. Their separation was achieved on a Zorbax Eclipse XDB C18 (100×4.6mm, 3.5μm) column using methanol and 0.1% acetic acid (65:35, v/v) as the mobile phase within 4.0min. Detection and quantitation were done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions, m/z 124.1→81.1,m/z 125.0→80.9 and m/z 141.0→81.0 for PZA, PA and 5-OH PA respectively in the positive ionization mode. All the analytes were baseline resolved with a resolution factor of 3.3 and 6.4 between PZA and its metabolites, PA and 5-OH PA respectively. The calibration curves were linear from 0.100-30.0μg/mL, 0.03-9.00μg/mL and 0.002-0.600μg/mL for PZA, PA and 5-OH PA respectively with r(2)≥0.9980 for all the analytes. The intra-batch and inter-batch accuracy and precision (% CV) across quality controls varied from 93.5-106.7% and 1.10-4.57 respectively for all the analytes. The mean extraction recovery of PZA, PA and 5-OH PA was 83.7%, 89.2% and 80.8% respectively, which was consistent at higher as well as lower concentration levels. The% change in the stability of analytes under different storage conditions ranged -6.7 to 7.1 for all the analytes. The method was applied to assess the comparative bioavailability of a 500mg PZA test and reference formulation in healthy subjects. The assay reproducibility was also tested by reanalysis of 22 incurred subject samples.

Challenges in optimizing sample preparation and LC‐MS/MS conditions for the analysis of carglumic acid, an N‐acetyl glutamate derivative in human plasma

This paper describes a systematic approach to overcoming challenges in developing a robust and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for reliable and precise determination of carglumic acid in human plasma. Sample extraction was tested on several reversed-phase solid-phase extraction (SPE) sorbents with different chemistries, such as hydrophobic C18, hydrophilic-lipophilic balance, and mixed-mode cation and anion exchange. The best recovery under the optimized extraction conditions was obtained with Oasis MAX (30 mg, 1cc) mixed-mode anion exchange (~ 50%) cartridge, compared to other sorbents from 100 μL plasma sample. Complete analytical separation of carglumic acid and carglumic acid-13C5 15N as an internal standard (IS) from endogenous plasma components was achieved on ACE 5CN (150 × 4.6 mm, 5 µm) column under isocratic conditions using acetonitrile:methanol (50:50, v/v) - 0.1% acetic acid in water [80:20, v/v] as the mobile phase. The deprotonated precursor → product ion transitions for carglumic acid (189/146) and IS (195/152) were monitored in the negative ionization mode on a triple quadrupole mass spectrometer. The regression curves were linear over a concentration range of 6.00-6000 ng/mL (r(2) ≥ 0.9987). Matrix effect was evaluated in terms of IS-normalized matrix factors, which ranged from 0.95 to 1.01 across four quality control levels. Intra- and inter-batch accuracy and precision, and the stability of carglumic acid in spiked plasma samples were assessed under different conditions. The method was applied to assess the pharmacokinetics of 100 mg/kg body weight carglumic acid in a healthy Indian subject.

Quantification of metronidazole in human plasma using a highly sensitive and rugged LC-MS/MS method for a bioequivalence study

A highly sensitive, selective and rugged method has been described for the quantification of metronidazole (MTZ) in human plasma by liquid chromatography-tandem mass spectrometry using metronidazole-d4 as the internal standard (IS). The analyte and the IS were extracted from 100 μL plasma by liquid-liquid extraction. The clear samples obtained were chromatographed on an ACE C18 (100 × 4.6 mm, 5 μm) column using acetonitrile and 10.0 mm ammonium formate in water, pH 4.00 (80:20, v/v) as the mobile phase. A triple quadrupole mass spectrometer system equipped with turbo ion spray source and operated in multiple reaction monitoring mode was used for the detection and quantification of MTZ. The calibration range was established from 0.01 to 10.0 μg/mL. The results of validation testing for precision and accuracy, selectivity, matrix effects, recovery and stability complied with current bioanalytical guidelines. A run time of 3.0 min permitted analysis of more than 300 samples in a day. The method was applied to a bioequivalence study with 250 mg MTZ tablet formulation in 24 healthy Indian males.

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.

Vibrational dynamics of some amorphous and quasicrystalline alloys

Inelastic nuclear resonance scattering has been used to get partial vibrational density of states (PVDOS) of iron in two classes of systems: (i) quasicrystalline Al 63.5 Cu 24 Fe 12.5 , its rhombohedral approximant and Al 70 Cu 20 Fe 10 crystalline (tetragonal) phase and (ii) amorphous Fe 80 B 20 and its crystalline counterpart. PVDOS in quasicrystalline phase consists of a rather broad maximum at 28 meV with a small hump at 21 meV, and is somewhat similar to that in crystalline Al 70 Cu 20 Fe 10 . Results suggest some similarity in the iron environment in quasicrystalline, approximant and crystalline phases. Amorphous Fe 80 B 20 exhibits an excess density of states as compared to the crystalline phase in the energy range 4-21 meV, which may be attributed to the boson peak.