Chemiluminescence determination of melamine with Luminol-K3Fe(CN)6 system

A sensitive chemiluminescence (CL) method was developed for determining melamine in urine and plasma samples based on the fact that melamine can remarkably enhance the chemiluminescence of Luminol-K₃ Fe(CN)₆ system in alkaline medium. The determination conditions were optimized. Under optimum conditions, the chemiluminescence intensity had a good linear relationship with melamine in the range of 9.0 × 10^?9 – 7.0 × 10^?6 g/mL with a correlation coefficient of 0.9992. The detection limits (3σ) were 3.54 ng/mL for urine sample and 6.58 ng/mL for plasma sample. The average recoveries of melamine were 102.6% for urine sample and 95.1% for plasma sample. Melamine in samples was extracted with liquid-liquid extraction procedures and the assay results coincided very well with that determined with flow injection chemiluminescence method. The method provides a reproducible and stable approach for sensitive detection and quantification of melamine in urine and plasma samples.     

Cathodic adsorptive stripping voltammetry of an anti-emetic agent Granisetron in pharmaceutical formulation and biological matrix

Granisetron showed one well-defined reduction peak at Hanging Mercury Drop Electrode (HMDE) in the potential range from ?1.3 to ?1.5 V due to reduction of C=N bond. Solid-phase extraction technique was employed for extraction of Granisetron from spiked human plasma. Granisetron showed peak current enhancement of 4.45% at square-wave voltammetry and 5.33% at cyclic voltammetry as compared with the non stripping techniques. The proposed voltammetric method allowed quantification of Granisetron in pharmaceutical formulation over the target concentration range of 50–200 ng/mL with detection limit 13.63 ng/mL, whereas in human plasma 50–225 ng/mL with detection limit 11.75 ng/mL.     

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.     

Dispersive liquid-liquid microextraction combined with high-performance liquid chromatography for the determination of clozapine and chlorpromazine in urine

A simple method has been proposed for the determination of clozapine (CLZ) and chlorpromazine (CPZ) in human urine by dispersive liquid-liquid microextraction (DLLME) in combination with high-performance liquid chromatography-ultraviolet detector (HPLC-UV). All important variables influencing the extraction efficiency, such as pH, types of the extraction solvent and the disperser solvent and their volume, ionic strength and centrifugation time were investigated and optimized. Under the optimal conditions, the limit of detection (LODs) and quantification (LOQs) of the method were 13 and 39 ng/mL for CLZ, and 2 and 6 ng/mL for CPZ, respectively. The relative standard deviations (RSDs) of the targets were less than 5.1% (C=0.100 μg/mL, n=9). Good linear behaviors over the tested concentration ranges were obtained with the values of R2>0.999 for the targets. The absolute extraction efficiencies of CLZ and CPZ from the spiked blank urine samples were 98.3% and 97.8%, respectively. The applicability of the technique was validated by analyzing urine samples and the mean recoveries for spiked urine samples ranged from 93.3% to 105.0%. The method was successfully applied for the determination of CLZ and CPZ in real human urine.     

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.     

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.     

Simultaneous determination of pioglitazone and candesartan in human plasma by LC-MS/MS and its application to a human pharmacokinetic study

A simple and rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and fully validated for simultaneous quantification of pioglitazone and candesartan in human plasma. Irbesartan was used as an internal standard. The analytes were extracted from human plasma samples by solid-phase extraction technique using a Strata-X 33 μm polymeric sorbent. The reconstituted samples were chromatographed on a C₁₈ column by using a 80:20 (v/v) mixture of acetonitrile and 0.1% formic acid as the mobile phase at a flow rate of 0.8 mL/min. The calibration curves obtained were linear (r≥0.99) over the concentration range of 15–3000 ng/mL for pioglitazone and 5–608 ng/mL for candesartan. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. A run time of 2.7 min for each sample made it possible to analyze more than 300 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.     

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.     

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.     

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.     

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.     

Sensitive determination of 4-O-methylhonokiol in rabbit plasma by high performance liquid chromatography and application to its pharmacokinetic investigation

A novel high Performance liquid Chromatographie method was developed for the determination of 4-O-methylhonokiol in rabbit plasma and was applied to its pharmacokinetic investigation. Plasma samples were treated by one-fold volume of methanol and acetonitrile to remove the interference proteins. A reverse phase column of SHIM-PACK VP-ODS (150 mm × 4.6 mm, 5.0 Mm) was used to separate 4-O-methylhonokiol in the plasma samples. The detection limit of 4-O-methylhonokiol was 0.2 μg/L and the linear ränge was 0.012 – 1.536 μg/L. The good extraction recoveries were obtained for the spiked samples (84.7%, 89.3% and 87.7% for low, middle and high concentrations of added Standards, respectively). The relative standard deviation of intra-day and inter-day precisions was in the ränge from 0.6% to 13.5%. The pharmacokinetic study of 4-O-methylhonokiol was made and the results from the plasma-concentration curve of 4-O-methylhonokiol showed a two-apartment open model. This work developed a sensitive, stable and rapid HPLC method for the determination of 4-O-methylhonokiol and the developed method has been successfully applied to a pharmacokinetic study of 4-O-methylhonokiol.     

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 and pharmacokinetic study of catechin in rat plasma by HPLC

A high performance liquid chromatographic method was developed and validated for the quantitative determination of catechin in rat plasma and its pharmacokinetic study after intragastric administration of Catechu and Xiongdanjiangre Wan into SD rats. Plasma samples were prepared by protein precipitation using methanol–5% aqueous zinc sulfate (70:30, v/v) as precipitant. Chromatographic separation was achieved on Hypersil C₁₈ column (250 mm×4.6 mm, 10 μm) with acetonitrile–water–triethylamine (6:94:0.3, v/v/v, pH 4.0±0.1, adjusted with phosphoric acid) as mobile phase, followed by a UV detection at 207 nm. Good linearity was obtained over the range of 0.143–7.15 mg/L of catechin, with correlation coefficient of 0.9992. The method was simple, sensitive, accurate and reproducible and has been successfully applied to the pharmacokinetic study of catechin in rat plasma.     

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 µL of human plasma. The chromatographic analysis was done on Acquity UPLC C18 (100 mm×2.1 mm, 1.7 µm) 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.     

Voltammetric quantification of anti-hepatitis drug Adefovir in biological matrix and pharmaceutical formulation

Electrochemical reduction behavior of Adefovir was studied using Hanging Mercury Drop Electrode (HMDE) in Britton–Robinson (BR) buffer solution. Voltammetric study showed one well-defined reduction peak in the potential range ?1.2 to ?1.4 V (vs. Ag/AgCl) due to reduction of C=N bond of the imidazole ring. Solid-phase extraction and protein-precipitation techniques were employed for extraction of Adefovir from human plasma. The proposed method allows quantification of Adefovir in human plasma over the concentration range 0.50–5.00 μg/mL with the detection limit 0.17 μg/mL, whereas in pharmaceutical formulation 0.25–2.25 μg/mL with the detection limit 0.08 μg/mL.     

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.     

The clinical value of enzyme-multiplied immunoassay technique monitoring the plasma concentrations of cyclosporine A after renal transplantation

The feasibility and the clinical value of the enzyme-multiplied immunoassay technique (EMIT) monitoring of blood concentrations of cyclosporine A (CsA) in patients treated with CsA were investigated after kidney transplantation. The validation method was performed to the EMIT determination of CsA blood concentration, the CsA whole blood ‘trough concentrations (C₀) of patients in different time periods after renal transplantation were monitored, and combined with the clinical complications, the statistical results were analyzed and compared. EMIT was precise, accurate and stable, also with a high quality control. The mean postoperative blood concentration of CsA was as follows: <1 month, (281.4 ± 57.9)ng/mL; 2 – 3 months, (264.5 ± 41. 2)ng/mL; 4 – 5 months, (236.4 ± 38. 9)ng/mL; 6 – 12 months, (206.5 ± 32.6)ng/mL; >12 months, (185.6 ± 28.1)ng/mL. The toxic reaction rate of CsA blood concentration within the recommended therapeutic concentration was 14. 1%, significantly lower than that of the none-recommended dose group (37.2%) (P < 0.05); the transplantation rejection rate was 4.4%, significantly lower than that of the none-recommended dose group (22.5%) (P < 0.05). Using EMIT to monitor the blood concentration of CsA as the routine laboratory method is feasible, and is able to reduce the CsA toxicity and rejection significantly, leading to achieving the desired therapeutic effect.     

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.