Improved simultaneous quantitation of candesartan and hydrochlorthiazide in human plasma by UPLC–MS/MS and its application in bioequivalence studies

A validated ultra-performance liquid chromatography mass spectrometric method (UPLC–MS/MS) was used for the simultaneous quantitation of candesartan (CN) and hydrochlorothiazide (HCT) in human plasma. The analysis was performed on UPLC–MS/MS system using turbo ion spray interface. Negative ions were measured in multiple reaction monitoring (MRM) mode. The analytes were extracted using a liquid–liquid extraction (LLE) method by using 0.1 mL of plasma volume. The lower limit of quantitation for CN and HCT was 1.00 ng/mL whereas the upper limit of quantitation was 499.15 ng/mL and 601.61 ng/mL for CN and HCT respectively. CN d₄ and HCT-¹³Cd₂ were used as the internal standards for CN and HCT respectively. The chromatography was achieved within 2.0 min run time using a C18 Phenomenex, Gemini NX (100 mm×4.6 mm, 5 µm) column with organic mixture:buffer solution (80:20, v/v) at a flow rate of 0.800 mL/min. The method has been successfully applied to establish the bioequivalence of candesartan cilexetil (CNC) and HCT immediate release tablets with reference product in human subjects.     

High performance liquid chromatography mass spectrometric method for the simultaneous quantification of pravastatin and aspirin in human plasma: Pharmacokinetic application

A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay method has been developed and fully validated for the simultaneous quantification of pravastatin and aspirin in human plasma. Furosemide was used as an internal standard. Analytes and the internal standard were extracted from human plasma by liquid–liquid extraction technique using methyl tertiary butyl ether. The reconstituted samples were chromatographed on a Zorbax SB-C₁₈ column by using a mixture of 5 mM ammonium acetate buffer and acetonitrile (20:80, v/v) as the mobile phase at a flow rate of 0.8 mL/min. The calibration curve obtained was linear (r≥0.99) over the concentration range of 0.50–600.29 ng/mL for pravastatin and 20.07–2012.00 ng/mL for aspirin. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.0 min for each sample made it possible to analyze more than 400 human plasma samples per day. The proposed method was found to be applicable to clinical studies.     

Simultaneous determination of telmisartan and amlodipine in human plasma by LC–MS/MS and its application in a human pharmacokinetic study

A rapid and sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay method has been developed and fully validated for the simultaneous quantification of telmisartan and amlodipine in human plasma. Carbamazepine was used as an internal standard. Analytes and the internal standard were extracted from human plasma by solid-phase extraction technique using Waters Oasis^® HLB 1 cm³ (30 mg) extraction cartridge. The reconstituted samples were chromatographed on a Hypurity advance C₁₈ column (50 mm×4.6 mm, 5 μm) using a mixture of acetonitrile–5 mM ammonium acetate buffer (pH-4.0) (50:50, v/v) as the mobile phase at a flow rate of 0.8 mL/min. The calibration curve obtained was linear (r≥0.99) over the concentration range of 2.01–400.06 ng/mL for telmisartan and 0.05–10.01 ng/mL for amlodipine. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. A run time of 2.5 min for each sample made it possible to analyze more than 400 human plasma samples per day. The proposed method was found to be applicable to clinical studies.     

Development and validation of RP-HPLC method for estimation of eplerenone in spiked human plasma

A rapid and simple high performance liquid chromatography (HPLC) method with a UV detection (241 nm) was developed and validated for estimation of eplerenone from spiked human plasma. The analyte and the internal standard (valdecoxib) were extracted with a mixture of dichloromethane and diethyl ether. The chromatographic separation was performed on a HiQSil C-18HS column (250 mm×4.6 mm, 5 μm) with a mobile phase consisting of acetonitrile:water (50:50, v/v) at flow rate of 1 mL/min. The calibration curve was linear in the range 100–3200 ng/mL and the heteroscedasticity was minimized by using weighted least squares regression with weighting factor 1/X.     

Chromatographic behavior of co-eluted plasma compounds and effect on screening of drugs by APCI-LC–MS(/MS): Applications to selected cardiovascular drugs

Chromatographic behavior of co-eluted compounds from un-extracted drug-free plasma samples was studied by LC–MS and LC–MS/MS with positive APCI. Under soft gradient, total ion chromatogram (TIC) consisted of two major peaks separated by a constant lower intensity region. Early peak (0.15–0.4 min) belongs to polar plasma compounds and consisted of smaller mass ions (m/z<250); late peak (3.6–4.6 min) belongs to thermally unstable phospholipids and consisted of fragments with m/z<300. Late peak is more sensitive to variations in chromatographic and MS parameters. Screening of most targeted cardiovascular drugs at levels lower than 50 ng/mL has been possible by LC–MS for drugs with retention factors larger than three. Matrix effects and recovery, at 20 and 200 ng/mL, were evaluated for spiked plasma samples with 15 cardiovascular drugs, by MRM–LC–MS/MS. Average recoveries were above 90% and matrix effects expressed as percent matrix factor (% MF) were above 100%, indicating enhancement character for APCI. Large uncertainties were significant for drugs with smaller masses (m/z<250) and retention factors lower than two.     

Validated LC–MS/MS method for simultaneous determination of SIM and its acid form in human plasma and cell lysate: Pharmacokinetic application

Simvastatin (SIM) is a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor widely used in hyperlipidemia therapy. SIM has recently been studied for its anticancer activity at doses higher than those used for the hyperlipidemia therapy. This prompted us to study the pharmacokinetics of high-dose SIM in cancer patients. For this purpose, an LC–MS/MS method was developed to measure SIM and its acid form (SIMA) in plasma and peripheral blood mononuclear cells (PBMCs) obtained from patients. Chromatographic analyte separation was carried out on a reverse-phase column using 75:25 (% v/v) acetonitrile:ammonium acetate (0.1 M, pH 5.0) mobile phase. Detection was performed on a triple quadrupole mass spectrometer, equipped with a turbo ion spray source and operated in positive ionization mode. The assay was linear over a range 2.5–500 ng/mL for SIM and 5–500 ng/mL for SIMA in plasma and 2.5–250 ng/mL for SIM and 5–250 ng/mL for SIMA in cell lysate. Recovery was >58% for SIM and >75% for SIMA in both plasma and cell lysate. SIM and SIMA were stable in plasma, cell lysate and the reconstitution solution. This method was successfully applied for the determination of SIM and SIMA in plasma and PBMCs samples collected in the pharmacokinetic study of high-dose SIM in cancer patients.     

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 spectrometry (LC–ESI–MS/MS) method has been developed for separation and quantification of metoprolol enantiomers on a chiral Lux Amylose-2 (250 mm×4.6 mm, 5 μm) column. Solid phase extraction of (S)-(−)- and (R)-(+)-metoprolol and rac-metoprolol-d6 as an internal standard (IS) was achieved on Lichrosep DVB HL cartridges employing 200 μL 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 different chromatographic methods developed for stereoselective analysis of metoprolol in biological matrices.     

Determination of metabolite of nicergoline in human plasma by high-performance liquid chromatography and its application in pharmacokinetic studies

A fast, simple and sensitive high performance liquid chromatographic (HPLC) method has been developed for determination of 10α-methoxy-6-methyl ergoline-8β-methanol (MDL, a main metabolite of nicergoline) in human plasma. One-step liquid–liquid extraction (LLE) with diethyl ether was employed as the sample preparation method. Tizanidine hydrochloride was selected as the internal standard (IS). Analysis was carried out on a Diamonsil ODS column (150 mm×4.6 mm, 5 μm) using acetonitrile–ammonium acetate (0.1 mol/L) (15/85, v/v) as mobile phase at detection wavelength of 224 nm. The calibration curves were linear over the range of 2.288–73.2 ng/mL with a lower limit of quantitation (LLOQ) of 2.288 ng/mL. The intra- and inter-day precision values were below 13% and the recoveries were from 74.47% to 83.20% at three quality control levels. The method herein described was successfully applied in a randomized crossover bioequivalence study of two different nicergoline preparations after administration of 30 mg in 20 healthy volunteers.     

Quantification of desloratadine in human plasma by LC-ESI-MS/MS and application to a pharmacokinetic study

A simple, sensitive, and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of desloratadine (DL) in human plasma using desloratadine-d5 (DLD5) as an internal standard (IS). Chromatographic separation was performed using an Xbridge C18 column (50 mm×4.6 mm, 5 μm) with an isocratic mobile phase composed of 10 mM ammonium formate: methanol (20:80, v/v), at a flow rate of 0.7 mL/min. DL and DLD5 were detected with proton adducts at m/z 311.2→259.2 and 316.2→264.3 in multiple reaction monitoring (MRM) positive modes, respectively. Liquid–liquid extraction (LLE) method was used to extract the drug and the IS. The method was validated over a linear concentration range of 5.0–5000.0 pg/mL with a correlation coefficient of (r²)≥0.9994. This method demonstrated intra- and inter-day precision within 0.7–2.0% and 0.7–2.7%, and an accuracy within 101.4–102.4%, and 99.5–104.8%. DL was found to be stable throughout the freeze–thaw cycles, bench-top, and postoperative stability studies. This method was successfully applied in the analysis of plasma samples following oral administration of DL (5 mg) in 35 healthy Indian male human volunteers under fasting conditions.     

LC-MS determination and pharmacokinetic study of salidroside in rat plasma after oral administration of suspensions of traditional Chinese medicine Erzhi Wan and Fructus Ligustri lucidi

A simple, rapid and sensitive liquid chromatography-mass spectrometry (LC-MS) method was developed for the determination of salidroside in rat plasma and study of its pharmacokinetics after oral administration of suspension of Erzhi Wan and Fructus Ligustri lucidi into Wistar rats. Plasma sample of 200 μL was extracted with acetic ether-isopropanol (2:1) and the extraction was performed on a Kromasil C₁₈ column (150 mm × 4. 6 mm, 5 μm) with the mobile phase of methanol-water (41:59, v/v) within a run time of 6.0 min. The analyte was monitored with positive electrospray ionization (ESI) by selected ion monitoring (SIM) mode. The target ions were m/z 323.05 for salidroside and m/z 411.05 for internal Standard (IS) geniposide. A good linear relationship was obtained over the range of 5.0–500.0 ng/mL and the lower limit of quantification was 5.0 ng/mL. The validated method was successfully applied to the pharmacokinetic study of salidroside in rat plasma after oral administration of suspension of Erzhi Wan and Fructus Ligustri lucidi.     

Ultra-performance liquid chromatography electrospray ionization–tandem mass spectrometry method for the simultaneous determination of itraconazole and hydroxy itraconazole in human plasma

A highly sensitive, selective, and precise ultra-performance liquid chromatography tandem mass spectrometry method was developed and validated for simultaneous quantification of itraconazole and hydroxy itraconazole in human plasma by a single liquid–liquid extraction step. The precursor to product ion transitions of m/z 705.3/392.3, m/z 721.2/408.3 and m/z 708.2/435.4 were used to detect and quantify itraconazole, hydroxy itraconazole and itraconazole-d3 respectively. The lower limit of quantitation was found to be 0.500 ng/mL for itraconazole and 1.00 ng/mL for hydroxy itraconazole. The mean recoveries for itraconazole and hydroxy itraconazole were found to be 100.045% and 100.021%, respectively. This developed method with a chromatographic run time of 2.0 min was successfully applied to a bioequivalence study of 100 mg itraconazole capsule.     

Simultaneous determination of ezetimibe and simvastatin in rat plasma by stable-isotope dilution LC-ESI–MS/MS and its application to a pharmacokinetic study

A simple, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed for simultaneous quantification of ezetimibe and simvastatin in rat plasma. The deuterium isotopes: ezetimibe d₄ and simvastatin d₆ were used as internal standards for ezetimibe and simvastatin, respectively. MS/MS detection involved a switch of electron spray ionization mode from negative to positive at retention time 3.01 min. Samples were extracted from plasma by liquid–liquid extraction using tertiary butyl methyl ether. Chromatographic separation was achieved with Agilent Eclipse XBD-C₁₈ column using mobile phase that consisted of a mixture of ammonium acetate (pH4.5; 10 mM)–acetonitrile (25:75 v/v). The method was linear and validated over the concentration range of 0.2–40.0 ng/mL for simvastatin and 0.05–15.0 ng/mL for ezetimibe. The transitions selected were m/z 408.3→271.1 and m/z 412.0→275.10 for ezetimibe and ezetimibe d₄, and m/z 419.30→285.20 and m/z 425.40→199.20 for simvastatin and simvastatin d₆. Intra- and inter-batch precisions for ezetimibe were 1.6–14.8% and 2.1–13.4%; and for simvastatin 0.94–9.56% and 0.79–12%, respectively. The proposed method was sensitive, selective, precise and accurate for the quantification of ezetimibe and simvastatin simultaneously in rat plasma. The method was successfully applied to a pharmacokinetic study by oral co-administration of ezetimibe and simvastatin in SD rats.     

Analysis of in vivo absorption of didanosine tablets in male adult dogs by HPLC

Didanosine is an effective antiviral drug in untreated and antiretroviral therapy-experienced patients with Human Immunodeficiency Virus (HIV). An automated system using on-line solid extraction and High Performance Liquid Chromatography (HPLC) with ultraviolet (UV) detection was developed and validated for pharmacokinetic analysis of didanosine in dog plasma. Modifications were introduced on a previous methodology for simultaneous analysis of antiretroviral drugs in human plasma. Extraction was carried out on C18 cartridges, with high extraction yield as stationary phase, whereas mobile phase consisted of a mixture of 0.02 M potassium phosphate buffer, acetonitrile (KH₂PO₄: acetonitrile: 96:4, v/v) and 0.5% (w/v) of heptane sulphonic acid. The pH was adjusted to 6.5 with triethylamine. All samples and standard solutions were chromatographed at 28 °C. For an isocratic run, the flux was 1.0 mL/min, detection was at 250 nm and injected volume was 20 μL. The method was selective and linear for concentrations between 50 and 5000 ng/mL. Drug stability data ranged from 96% to 98%, and limit of quantification was 25 ng/mL. Extraction yield was up to 95%. Drug stability in dog plasma was kept frozen at ?20 °C for one month after three freeze–thaw cycles, and for 24 h after processing in the auto sampler. Assay was successfully applied to measure didanosine concentrations in plasma dogs.     

Quantification of sibutramine and its two metabolites in human plasma by LC–ESI-MS/MS and its application in a bioequivalence study

Obesity can be considered as a chronic illness of epidemic proportion and its incidents have increased exponentially in recent years. The use of anti-obesity drugs such as sibutramine is somewhat helpful. There is a need to quantify such drugs in biological samples, which is generally quite difficult. In this report, we developed and validated a simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of sibutramine (SB) and its two metabolites N-des methyl sibutramine (DSB) and N-di desmethyl sibutramine (DDSB) in human plasma. Zorbax SB-C18 (4.6 mm×75 mm, 3.5 μm, 80 Å) analytical column and 5 mM ammonium formate:acetonitrile (10:90, v/v) mobile phase were used for chromatographic separation of SB, DSB and DDSB. Multiple reaction monitoring (MRM) in the positive mode was used to detect SB, DSB and DDSB at m/z 280.3/124.9, 266.3/125.3 and 252.2/124.9, respectively. Liquid–liquid extraction was used for the extraction of analytes and internal standard from human plasma. This method was validated over a linear concentration range of 10.0–10,000.0 pg/mL for SB, DSB and DDSB with correlation coefficients (r) of ≥0.9997. The drug and the two metabolites were stable in plasma samples. The validated method was successfully applied in a bioequivalence and pharmacokinetic study with human volunteers under fasting condition.     

Validation and application by HPLC for simultaneous determination of vitexin-2″-O-glucoside,vitexin-2″-O-rhamnoside,rutin, vitexin,and hyperoside

A simple, precise, and rapid high-performance liquid chromatographic method was developed and validated for the simultaneous determination of vitexin-2″-O-glucoside, vitexin-2″-O-rhamnoside, rutin, vitexin, and hyperoside. The HPLC separation was performed using a Shim-pack VP-ODS C₁₈ column (250 mm×4.6 mm i.d., 5 μm) with the isocratic mobile phase consisting of tetrahydrofuran/ acetonitrile/0.05% phosphoric acid solution (20:3:77, v/v/v), and the flow rate was set at 1.0 mL/min. UV detection was carried out at a wavelength of 360 nm and the whole analysis took 25 min. The method was linear in the range of 4.12–206.00 μg/mL for vitexin-2″-O-glucoside, 4.05–202.50 μg/mL for vitexin-2″-O-rhamnoside, 1.64–82.00 μg/mL for rutin, 1.74–87.00 μg/mL for vitexin, and 1.41–70.60 μg/mL for hyperoside with the correlation coefficient for each analyte more than 0.998. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.6 and 2 ng for vitexin-2″-O-glucoside, 0.6 and 2 ng for vitexin-2″-O-rhamnoside, 0.3 and 1 ng for rutin, 1 and 3 ng for vitexin, and 0.5 and 2 ng for hyperoside, respectively. Intra- and inter-day precision and accuracy (RSD) were less than 3%. The developed HPLC method was successfully applied to the analysis of five flavonoids in hawthorn leaves, hawthorn fruits, and the preparations containing hawthorn leaves or fruits.     

Development and validation of analytical method for the estimation of nateglinide in rabbit plasma

Nateglinide has been widely used in the treatment of type-2 diabetics as an insulin secretogoga. A reliable, rapid, simple and sensitive reversed-phase high performance liquid chromatography (RP-HPLC) method was developed and validated for determination of nateglinide in rabbit plasma. The method was developed on Hypersil BDSC-18 column (250 mm×4.6 mm, 5 mm) using a mobile phase of 10 mM phosphate buffer (pH 2.5) and acetonitrile (35:65, v/v). The elute was monitored with the UV–vis detector at 210 nm with a flow rate of 1 mL/min. Calibration curve was linear over the concentration range of 25–2000 ng/mL. The retention times of nateglinide and internal standard (gliclazide) were 9.608 min and 11.821 min respectively. The developed RP-HPLC method can be successfully applied to the quantitative pharmacokinetic parameters determination of nateglinide in rabbit model.     

Development and validation of analytical method for the estimation of lamivudine in rabbit plasma

Lamivudine has been widely used in the treatment of HIV disease. A reliable, sensitive reversed phase high performance liquid chromatography (RP-HPLC) method was developed and validated for lamivudine in rabbit plasma. The method was developed on Hypersil BDS C-18 column (250 mm×4.6 mm, 5 μm) using a mobile phase of 0.25% Triethylamine buffer (pH 3.0): acetonitrile (70:30, v/v). The efficient was monitored by UV detector at 256 nm. The total run time was 15 min with a flow rate of 1.0 mL/min. Calibration curve was linear over the concentration range of 25–2000 ng/mL. The retention times of lamivudine and internal standard (Nelfinavir) were 8.78 min and 10.86 min, respectively. The developed RP-HPLC method can be successfully applied for the quantitative pharmacokinetic parameters determination of lamivudine in rabbit model.     

Extraction and determination of trace amounts of chlorpromazine in biological fluids using magnetic solid phase extraction followed by HPLC

A simple, rapid and sensitive method termed as magnetic solid phase extraction (MSPE) combined with high-performance liquid chromatography-ultraviolet detector (HPLC-UV) has been proposed for the determination of trace amounts of chlorpromazine (CPZ) in water, urine and plasma samples. The separation and determination was performed on a C18 column under the optimal chromatographic conditions. Several factors influencing the extraction efficiency of CPZ, such as pH, surfactant and adsorbent amounts, ionic strength, extraction time, sample volume and desorption conditions, were studied and optimized. Under the optimal MSPE conditions, the extraction percentage of CPZ was 74%, 27% and 16% in water, urine and plasma samples, respectively. The limits of detection (LODs) of the proposed approach were 0.1, 5.0 and 10 ng/mL in water, urine and plasma samples, respectively. The relative standard deviations (RSDs) based on five replicate determinations at 10 ng/mL level of CPZ was 1.2%. Good linear behaviors over the investigated concentration ranges (0.25–300 ng/mL) with good coefficient of determination, R²>0.9998, were obtained. Good spike recoveries with relative errors less than 9.0% were obtained when applying the proposed method to water, urine and plasma samples.     

Precolumn derivatization LC–MS/MS method for the determination and pharmacokinetic study of glucosamine in human plasma and urine

A selective precolumn derivatization liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the determination of glucosamine in human plasma and urine has been developed and validated. Glucosamine was derivatized by o-phthalaldehyde/3-mercaptopropionic acid. Chromatographic separation was performed on a Phenomenex ODS column (150 mm×4.6 mm, 5 μm) using linear gradient elution by a mobile phase consisting of methanol (A), and an aqueous solution containing 0.2% ammonium acetate and 0.1% formic acid (B) at a flow rate of 1 mL/min. Tolterodine tartrate was used as the internal standard (IS). With protein precipitation by acetonitrile and then the simple one-step derivatization, a sensitive bio-assay was achieved with the lower limit of quantitation (LLOQ) as low as 12 ng/mL for plasma. The standard addition calibration curves suitable for clinical sample analysis showed good linearity over the range of 0.012–8.27 μg/mL in plasma and 1.80–84.1 μg/mL in urine. The fully validated method has been successfully applied to a pharmacokinetic study of compound glucosamine sulfate dispersible tablets in health Chinese volunteers receiving single oral doses at 500, 1000 and 1500 mg of glucosamine sulfate, as well as multiple oral doses of 500 mg t.i.d. for 7 consecutive days.     

Kinetics study of metaxalone degradation under hydrolytic,oxidative and thermal stress conditions using stability-indicating HPLC method

An isocratic stability indicating RP-HPLC–UV method is presented for the determination of metaxalone (MET) in the presence of its degradation products. The method uses Dr. Maisch C18 column (250 mm×4.6 mm, 5 μm) with mobile phase consisting of acetonitrile–potassium dihydrogen orthophosphate buffer with 4 mL of 0.4% triethyl amine (pH 3.0; 10 mM) (58:42, v/v) at a flow rate of 1.0 mL/min. pH of the buffer was adjusted with o-phosphoric acid. UV detection was performed at 225 nm. The method was validated for specificity, linearity, precision, accuracy, limit of detection, limit of quantification and robustness. The calibration plot was linear over the concentration range of 1–100 μg/mL having a correlation coefficient (r²) of 0.999. Limits of detection and quantification were 0.3 and 1 μg/mL, respectively. Intra-day and inter-day precision (% RSD) was 0.65 and 0.79 respectively. The proposed method was used to investigate the degradation kinetics of MET under different stress conditions employed. Degradation of MET followed a pseudo-first-order kinetics, and rate constant (K), time left for 50% potency (t_1/2), and time left for 90% potency (t₉₀) were calculated.