Sensitive and rapid determination of amantadine without derivatization in human plasma by LC–MS/MS for a bioequivalence study

A highly sensitive, rapid and rugged liquid chromatography-tandem mass spectrometw （LC-ESI-MS/MS） method was developed for reliable estimation of amantadine （AMD）, an antiviral drug in human plasma. The analyte and internal standard （IS）, amantadine-d6 （AMD-d6）, were extracted from 200μL plasma by solid phase extraction on Phenomenex Strata-X-C 33 ~t cartridges. Chromatography was performed on Synergi^TM Hydro-RP C18 （150 mm×4.6 mm, 4 μm） analytical column using a mixture of acetonitrile and 10 mM ammonium formate, pH 3.0 （80：20, v/v） as the mobile phase. Detection and quantitation was done by multiple reaction monitoring in the positive ionization mode for AMD （m/z 152.1→ 135.1 ） and IS （m/z 158.0 → 141.1） on a triple quadrupole mass spectrometer. The assay was linear in the concentration range of 0.50-500 ng/mL with correlation coefficient （r^2） 〉 0.9969. The limit of detection of the method was 0.18 ng/mL The intra-batch and inter-batch precisions were 〈 5.42% and the accuracy varied from 98.47% to 105.72%. The extraction recovery of amantadine was precise and quantitative in the range of 97.89%-100.28%. IS-normalized matrix factors for amantadine varied from 0.981 to 1.012. The stability of AMD in whole blood and plasma was evaluated under different conditions. The developed method was successfully applied for a bioequivalence study with 100 mg of AMD in 32 healthy volunteers. The reproducibility of the assay was determined by reanalysis of 134 subject samples.

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 C_(18)(50 mm×3.0 mm, 5 μm) column. The analytes were eluted within 2.0 min using acetonitrile-5.0 m M ammonium formate, p H 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/m L 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.

Application of an LC–MS/MS method for the analysis of amlodipine,valsartan and hydrochlorothiazide in polypill for a bioequivalence study

A sensitive and selective method has been proposed for the simultaneous determination of amlodipine(AML),valsartan(VAL) and hydrochlorothiazide(HCTZ) in human plasma by liquid chromatography–tandem mass spectrometry(LC–MS/MS). The analytes and their deuterated analogs were quantitatively extracted from100 μL human plasma by solid phase extraction on Oasis HLB cartridges. The chromatographic separation of the analytes was achieved on a Chromolith RP18 e(100 mm × 4.6 mm) analytical column within 2.5 min. The resolution factor between AML and VAL, AML and HCTZ, and VAL and HCTZ was 2.9, 1.5 and 1.4, respectively,under isocratic conditions. The method was validated over a dynamic concentration range of 0.02–20.0 ng/m L for AML, 5.00–10,000 ng/m L for VAL and 0.20–200 ng/m L for HCTZ. Ion-suppression/enhancement effects were investigated by post-column infusion technique. The mean IS-normalized matrix factors for AML, VAL and HCTZ were 0.992, 0.994 and 0.998, respectively. The intra-batch and inter-batch precision(% CV) across quality control levels was ≤ 5.56% and the recovery was in the range of 93.4%–99.6% for all the analytes. The method was successfully applied to a bioequivalence study of 5 mg AML + 160 mg VAL + 12.5 mg HCTZ tablet formulation(test and reference) in 18 healthy Indian males under fasting. The mean log-transformed ratios of C max, AUC0–120 h and AUC0-inf and their 90% CIs were within 90.2%–102.1%. The assay reproducibility was demonstrated by reanalysis of 90 incurred samples.

Development of a sensitive and rapid method for quantitation of (S)-(-)- and (R)-(+)-metoprolol in human plasma by chiral LC–ESI–MS/MS

A selective, sensitive and high throughput liquid chromatography-tandem mass spectro-metry （LC-ESI-MS/MS） method has been developed for separation and quantification of metoprolol enantiomers on a chiral Lux Amylose-2 （250 mm＆#215;4.6 mm, 5 mm） column. Solid phase extraction of （S）-（-）- and （R）-（t）-metoprolol and rac-metoprolol-d6 as an internal standard （IS） was achieved on Lichrosep DVB HL cartridges employing 200 mL human plasma. Both the analytes were chromatographically separated with a resolution factor of 2.24 using 15 mM ammonium acetate in water, pH 5.0 and 0.1% （v/v） diethyl amine in acetonitrile （50：50, v/v） as the mobile phase within 7.0 min. The precursor-product ion transitions for the enantiomers and IS were monitored in the multiple reaction monitoring and positive ionization mode. The method was validated over the concentration range of 0.500-500 ng/mL for both the enantiomers. Matrix effect was assessed by post-column analyte infusion experiment and the mean extraction recovery was greater than 94.0% for both the enantiomers at all quality control levels. The stability of analytes was evaluated in plasma and whole blood under different storage conditions. The method was successfully applied to a clinical study in 14 healthy volunteers after oral administration of 200 mg metoprolol tablet under fasting conditions. The assay reproducibility is shown by reanalysis of 68 incurred samples. The suitability of the developed method was assessed in comparison with＆amp;nbsp;different chromatographic methods developed for stereoselective analysis of metoprolol in biological matrices.

Determination of lercanidipine in human plasma by an improved UPLC–MS/MS method for a bioequivalence study

An improved and reliable ultra-performance liquid chromatography/tandem mass spectrometry （UPLC- MS/MS） method has been developed and validated for the determination of lercanidipine in human plasma. Plasma samples with lercanidipine-d3 as an internal standard （IS） were prepared by solid phase extraction on Phenomenex Strata-X cartridges using 100 μL of human plasma. Chromatographic analysis was performed on UPLC BEH C18 （50 mm × 2.1 mm, 1.7 μm） column under isocratic conditions. Linear calibration curves were obtained over a wide dynamic concentration range of 0.010-20.0 ng/mL. Matrix effect was assessed by post-column infusion, post-extraction spiking and standard-line slope methods. The mean extraction recovery was 〉 94% for the analyte and IS. Inter-batch and intra-hatch precision （% CV） across five quality controls was 〈 5.8%. Bioequivalence study was performed with 36 healthy sub- jects after oral administration of 10 mg of lercanidipine and the assay reproducibility was evaluated by reanalysis of 133 incurred samples.

Simultaneous quantification of amiloride and hydrochlorothiazide in human plasma by liquid chromatography–tandem mass spectrometry

A selective, sensitive and precise assay based on solid phase extraction and liquid chromatography–tandem mass spectrometry(LC–MS/MS) was developed for the simultaneous determination of amiloride(AMI) and hydrochlorothiazide(HCTZ) in human plasma. Sample clean-up with 250 μL of plasma was done on Phenomenex Strata^(?)-X extraction cartridges using their labeled internal standards(AMI-15 N3 and HCTZ-13 C,d2). Chromatography was performed on Hypersil Gold C18(50 mm×3.0 mm, 5 μm) column using acetonitrile with 4.0 m M ammonium formate(p H 4.0, adjusted with 0.1% formic acid)(80:20, v/v) as the mobile phase. Detection was carried out on a triple quadrupole API 5500 mass spectrometer utilizing an electrospray ionization interface and operating in the positive ionization mode for AMI and negative ionization mode for HCTZ. Multiple reaction monitoring was used following the transitions at m/z 230.6/116.0, m/z233.6/116.0, m/z 296.0/204.9 and m/z 299.0/205.9 for AMI, AMI-15 N3, HCTZ and HCTZ-13 C,d2,respectively. Calibration curves were linear(r2≥0.9997) over the concentration range of 0.050–50.0 and0.50–500 ng/m L for AMI and HCTZ, respectively, with acceptable accuracy and precision. The signal-to-noise ratio at the limit of quantitation was ≥14 for both the analytes. The mean recovery of AMI and HCTZ from plasma was 89.0% and 98.7%, respectively. The IS-normalized matrix factors determined for matrix effect ranged from 0.971 to 1.024 for both the analytes. The validated LC–MS/MS method was successfully applied to a bioequivalence study using 5 mg AMI and 50 mg HCTZ fixed dose tablet formulation in 18 healthy Indian volunteers with good reproducibility.

Determination of asenapine in presence of its inactive metabolites in human plasma by LC-MS/MS

A highly selective and sensitive liquid chromatography-tandem mass spectrometry （LC-MS/MS） assay has been described for the determination of asenapine （ASE） in presence of its inactive metabolites N-desmethyl asenapine （DMA） and asenapine-N-glucuronide （ASG）. ASE, and ASE 13C-d3, used as internal standard （IS）, were extracted from 300 μL human plasma by a simple and precise liquid-liquid extraction procedure using methyl tert-butyl ether. Baseline separation of ASE from its inactive metabolites was achieved on Chromolith Performance RPse （100 mm ×4.6 mm） column using acetonitrile- 5.0 mM ammonium acetate-10% formic acid （90：10：0.1, v/v/v） within 4.5 min. Quantitation of ASE was done on a triple quadrupole mass spectrometer equipped with electrospray ionization in the positive mode. The protonated precursor to product ion transitions monitored for ASE and ASE 13C-d3 were m/z 286.1 → 166.0 and m/z 290.0 → 166.1, respectively. The limit of detection （LOD） and limit of quantitation （LOQ） of the method were 0.0025 ng/mL and 0.050 ng/mL respectively in a linear concentration range of 0.050-20.0 ng/mL for ASE. The intra-batch and inter-batch precision （% CV） and mean relative recovery across quality control levels were 〈 5.8% and 87.3%, respectively. Matrix effect, evaluated as IS-normalized matrix factor, ranged from 1.03 to 1.05. The stability of ASE under different storage conditions was ascertained in presence of the metabolites. The developed method is much simpler, matrix free, rapid and economical compared to the existing methods. The method was suc- cessfully used for a bioequivalence study of asenapine in healthy Indian subjects for the first time.

Determination of cilostazol and its active metabolite 3,4-dehydro cilostazol from small plasma volume by UPLC-MS/MS

A simple,rapid and sensitive ultra performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS） method has been developed for the simultaneous determination of cilostazol and its pharmacologically active metabolite 3,4-dehydro cilostazol in human plasma using deuterated analogs as internal standards（ISs）.Plasma samples were prepared using solid phase extraction and chromatographic separation was performed on UPLC BEH C18（50 mm × 2.1 mm.1.7 μm） column.The method was established over a concentration range of 0.5-1000 ng/mL for cilostazol and 0.5-500 ng/mL for 3.4-dehydro cilostazol.Intra- and inter-batch precision（%CV） and accuracy for the analytes were found within 0.93-1.88 and 98.8-101.7% for cilostazol and 0.91-2.79 and 98.0-102.7% for the metabolite respectively.The assay recovery was within 95-97% for both the analytes and internal standards.The method was successfully applied to support a bioequivalence study of 100 mg cilostazol in30 healthy subjects.

Determination of mycophenolic acid in human plasma by ultra performance liquid chromatography tandem mass spectrometry

A simple, sensitive and high throughput ultra performance liquid chromatography tandem mass spectrometry method has been developed for the determination of mycophenolic acid in human plasma. The method involved simple protein precipitation of MPA along with its deuterated analog as an internal standard （IS） from 50 mL of human plasma. The chromatographic analysis was done on Acquity UPLC C18 （100mm×2.1mm,1.7μm） column under isocratic conditions using acetonitrile and 10 mM ammonium formate, pH 3.00 （75：25, v/v） as the mobile phase. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for quantitation. In-source conversion of mycophenolic glucuronide metabolite to the parent drug was selectively controlled by suitable optimization of cone voltage, cone gas flow and desolvation temperature. The method was validated over a wide concentration range of 15-15000 ng/mL. The mean extraction recovery for the analyte and IS was 〉95%. Matrix effect expressed as matrix factors ranged from 0.97 to 1.02. The method was successfully applied to support a bioequivalence study of 500 mg mycophenolate mofetil tablet in 72 healthy subjects.

Liquid chromatography-tandem mass spectrometry method for simultaneous determination of albendazole and albendazole sulfoxide in human plasma for bioequivalence studies

An improved high performance liquid chromatography-tandem mass spectrometry（LC-MS/MS） method has been developed for sensitive and rapid determination of albendazole（ABZ） and its active metabolite,albendazole sulfoxide（ABZSO）,in the positive ionization mode.The method utilized solid phase extraction（SPE） for sample preparation of the analytes and their deuterated internal standards（ISs） from100 μL human plasma.The chromatography was carried out on Hypurity C_（18） column using acetonitrile-2.0 mM ammonium acetate,pH 5.0（80：20,v/v） as the mobile phase.The assay exhibited a linear response over the concentration range of 0.200-50.0 ng/mL for ABZ and 3.00-600 ng/mL for ABZSO.The recoveries of the analytes and ISs ranged from 86.03%-89.66%and 89.85%-98.94%,respectively.Matrix effect,expressed as IS-normalized matrix factors,ranged from 0.985 to 1.042 for the both analytes.The method was successfully applied for two separate studies in healthy subjects using single dose of 400 mg conventional tablets and 400 mg chewable ABZ tablets,respectively.

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 C_（18）（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/z286.2→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（r^2 ≥ 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 of2.5 mg tablet formulation to 16 healthy postmenopausal Indian women.The assay reproducibility was also established through incurred sample reanalysis（ISR） of 74 subject samples.

Highly sensitive LC–MS/MS method to estimate doxepin and its metabolite nordoxepin in human plasma for a bioequivalence study

A selective, sensitive and rugged liquid chromatography–tandem mass spectrometry(LC–MS/MS) assay has been developed for the simultaneous determination of doxepin(Dox) and its pharmacologically active metabolite, nordoxepin(NDox) in human plasma. The analytes and their internal standards(IS)were extracted from 500 m L of human plasma by liquid-liquid extraction using methyl tert-butyl ether.Chromatographic separation was achieved on Hypurity C8 column(100 mm ? 4.6 mm, 5 mm) using a mixture of acetonitrile-methanol(95:5, v/v) and 2.0 mM ammonium formate in 93:7(v/v) ratio. Detection was accomplished by tandem mass spectrometry in the positive ionization and multiple reaction monitoring acquisition mode. The protonated precursor to product ion transitions studied for Dox, NDox,and their corresponding ISs, propranolol and desipramine, were m/z 280.1-107.0, 266.0-107.0,260.1-116.1 and 267.1-72.1, respectively. A linear dynamic range of 15.0–3900 pg/mL for Dox and 5.00–1300 pg/mL for NDox was established with mean correlation coefficient(r2) of 0.9991 and 0.9993, respectively. The extraction recovery ranged from 86.6%–90.4% and 88.0%–99.1% for Dox and NDox, respectively. The intra-batch and inter-batch precision(% CV) across quality control levels was r 8.3% for both the analytes. Stability evaluated under different storage conditions showed no evidence of degradation and the % change in stability samples compared to nominal concentration ranged from 4.7% to12.3%. The method was successfully applied to a bioequivalence study of 6 mg doxepin hydrochloride orally disintegrating tablet in 41 healthy Indian subjects under fasting and fed conditions.

Simultaneous enantioseparation and simulation studies of atenolol,metoprolol and propranolol on Chiralpak^■IG column using supercritical fluid chromatography

Enantioseparation of threeβ-blockers,i.e.,atenolol,metoprolol and propranolol,was studied on amylose tris(3-chloro-5-methylphenylcarbamate)immobilized chiral stationary phase using supercritical fluid chromatography(SFC).The effect of organic modifiers(methanol,isopropanol and their mixture),column temperature and back pressure on chiral separation ofβ-blockers was evaluated.Optimum chromatographic separation with respect to resolution,retention,and analysis time was achieved using a mixture of CO_(2) and 0.1%isopropyl amine in isopropanol:methanol(50:50,V/V),in 75:25(V/V)ratio.Under the optimized conditions,the resolution factors(Rs)and separation factors(a)were greater than3.0 and 1.5,respectively.Further,with increase in temperature(25-45℃)and pressure(100-150 bars)there was corresponding decrease in retention factors(k),a and Rs.However,a reverse trend(a and Rs)was observed for atenolol with increase in temperature.The thermodynamic data from van’t Hoff plots revealed that the enantioseparation was enthalpy driven for metoprolol and propranolol while entropy driven for atenolol.To understand the mechanism of chiral recognition and the elution behavior of the enantiomers,molecular docking studies were performed.The binding energies obtained from simulation studies were in good agreement with the elution order found experimentally and also with the free energy values.The method was validated in the concentration range of 0.5-10μg/m L for all the enantiomers.The limit of detection and limit of quantitation ranged from 0.126 to 0.137μg/m L and 0.376-0.414μg/m L,respectively.The method was used successfully to analyze these drugs in pharmaceutical preparations.

An improved LC–MS/MS method for the quantification of alverine and para hydroxy alverine in human plasma for a bioequivalence study

A highly sensitive and selective high performance liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of alverine（ALV） and its active metabolite, para hydroxy alverine（PHA）, in human plasma. For sample preparation, solid phase extraction of analytes was performed on Phenomenex Strata-X cartridges using alverine-d5 as the internal standard. The analytes were separated on Symmetry Shield RP18（150 mm×3.9 mm, 5 μm） column with a mobile phase consisting of acetonitrile and10 mM ammonium formate（65：35, v/v）. Detection and quantitation was done by electrospray ionization mass spectrometry in the positive mode using multiple reaction monitoring. The assay method was fully validated over the concentration range of 15.0–15,000 pg/mL for ALV and 30.0–15,000 pg/mL for PHA. The intra-day and inter-day accuracy and precision（% CV） ranged from 94.00% to 96.00% and 0.48% to 4.15% for both the analytes. The mean recovery obtained for ALV and PHA was 80.59% and 81.26%, respectively. Matrix effect,expressed as IS-normalized matrix factor ranged from 0.982 to 1.009 for both the analytes. The application of the method was demonstrated for the specific analysis of ALV and PHA for a bioequivalence study in 52 healthy subjects using 120 mg ALV capsules. The assay reproducibility was also verified by reanalysis of 175 incurred subject samples.

Selective and rapid determination of raltegravir in human plasma by liquid chromatography–tandem mass spectrometry in the negative ionization mode

A selective and rapid high-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of raltegravir using raltegravir-d3 as an internal standard（IS）. The analyte and IS were extracted with methylene chloride and n-hexane solvent mixture from 100 mL human plasma. The chromatographic separation was achieved on a Chromolith RP-18 e endcapped C18（100 mm 4.6 mm） column in a run time of 2.0 min. Quantitation was performed in the negative ionization mode using the transitions of m/z 443.1-316.1 for raltegravir and m/z 446.1-319.0 for IS. The linearity of the method was established in the concentration range of 2.0–6000 ng/m L.The mean extraction recovery for raltegravir and IS was 92.6% and 91.8%, respectively, and the IS-normalized matrix factors for raltegravir ranged from 0.992 to 0.999. The application of this method was demonstrated by a bioequivalence study on 18 healthy subjects.

LC-MS/MS assay for olanzapine in human plasma and its application to a bioequivalence study

This paper describes a selective and sensitive assay for the determination of olanzapine(OLZ)in human plasma based on liquid chromatography-tandem mass spectrometry(LC-MS/MS).The analyte and quetiapine as internal standard(IS)were extracted from 200μL plasma via solid phase extraction on Waters Oasis HLB cartridges.Chromatographic separation was achieved on an ACE 5C18-300 column(100 mm × 4.6 mm,5μm)under isocratic conditions in a run time of 3.5 min.Mass spectrometric detection involved electrospray ionization in the positive ion mode followed by multiple reaction monitoring(MRM)of the transitions at m/z 313/256 for OLZ and m/z 384/253 for the IS.The assay was linear in the range 0.10-40.0 ng/mL with a lower limit of quantitation and limit of detection of 0.10 and 0.012 ng/mL,respectively.Intra-and inter-day precision(as coefficient of variation)and relative recovery were<5.0% and>90%,respectively.The method was succesfully applied to a bioequivalence study of 5 and 10 mg OLZ disintegrating tablets in 40 healthy Indian males with reproducibility by incurred sample reanalysis in the range-7.43 to 8.07%.