Flow injection chemiluminescence determination of loxoprofen and naproxen with the acidic permanganate-sulfite system

A novel flow injection chemiluminescence (CL) method for the determination of loxoprofen and naproxen was proposed based on the CL system of KMnO₄, and Na₂SO₃ in acid media. The CL intensity of KMnO₄-Na₂SO₃ was greatly enhaneed in the presence of loxoprofen and naproxen. The mechanism of the CL reaction was studied by the kinetic proecss and UV-vis absorption and the conditions were optimized. Under optimized conditions, the CL intensity was linear with loxoprofen and naproxen concentration in the range of 7.0 × 10^?8 – 1.0 × 10^?5 g/mL and 2.0 × 10^?7 – 4.0 × 10^?6 g/mL with the detection limit of 2.0 × 10^?8 g/mL and 3.0 × 10^?8 g/mL (S/N = 3), respectively. Thc relative standard deviations were 2.39% and 1.37% for 5.0 × 10^?7 g/mL naproxen and 5.0 × 10^?7 g/mL loxoprofen (n = 10), respectively. The proposed method was satisfactorily applied to thc determination of loxoprofen and naproxen in pharmaceutical preparations.     

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.     

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.     

Rapid europium-sensitized fluorescent determination of ulifloxacin,the active metabolite of prulifloxacin,in human serum and urine

A new fluorescent method was developed based on the ulifloxacin-europium (III)-sodium dodecylbenzene sulfonate system for the determination of ulifloxacin, the active metabolite of prulifloxacin. Sodium dodecylbenzene sulfonate formed a ternary complex with ulifloxacin-europium (III) and significantly enhaneed the characteristic fluorescence of europium (III). The enhanced fluorescence intensity showed a good linear relationship with the concentration of ulifloxacin in the range of 5.0 × 10^?8 – 2.0 × 10^?6M with a detection limit of 2.0 × 10^?10 M (3σ). This method is rapid and sensitive, and has been successfully applied to the determination of ulifloxacin in human urine and serum samples.     

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.     

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.     

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.     

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.     

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.     

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.     

Cleaning level acceptance criteria and HPLC-DAD method validation for the determination of Nabumetone residues on manufacturing equipment using swab sampling

Prevention of cross contamination with active pharmaceutical ingredients is crucial and requires special attention in pharmaceutical industries. Current method validation describes the determination of Nabumetone (NAB) residue on a stainless steel surface using swab sampling with a sensitive HPLC-DAD analysis. The acceptance limit was decided as 2 μg swab per 100 cm². Cotton swabs impregnated with extraction solution were used to determine residual drug content. Recoveries were 90.88%, 91.42%, and 92. 21% with RSD ranging from 2.2% to 3.88% at three concentration levels. Residual concentration was found to be linear in the range of 0.1–4.56 μg/mL, when estimated using a Phenomenex Luna C₁₈ (25 cm×5 μm×4.6 mm i.d.) column at 1.0 mL/min flow rate and 230 nm. The mobile phase consisted of a mixture of methanol:acetonitrile:water (55:30:15, v/v/v). The LOD and LOQ for NAB were found to be 0.05 and 0.16 μg/mL, respectively. The validated method was found to be simple, selective and sensitive for demonstration of cleaning validation of NAB residues on a stainless steel surface.     

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.     

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.     

Bio-analytical method development and validation of Rasagiline by high performance liquid chromatography tandem mass spectrometry detection and its application to pharmacokinetic study

The most suitable bio-analytical method based on liquid–liquid extraction has been developed and validated for quantification of Rasagiline in human plasma. Rasagiline-¹³C₃ mesylate was used as an internal standard for Rasagiline. Zorbax Eclipse Plus C18 (2.1 mm×50 mm, 3.5 μm) column provided chromatographic separation of analyte followed by detection with mass spectrometry. The method involved simple isocratic chromatographic condition and mass spectrometric detection in the positive ionization mode using an API-4000 system. The total run time was 3.0 min. The proposed method has been validated with the linear range of 5–12000 pg/mL for Rasagiline. The intra-run and inter-run precision values were within 1.3%–2.9% and 1.6%–2.2% respectively for Rasagiline. The overall recovery for Rasagiline and Rasagiline-¹³C₃ mesylate analog was 96.9% and 96.7% respectively. This validated method was successfully applied to the bioequivalence and pharmacokinetic study of human volunteers under fasting condition.     

Pharmacokinetic behavior of 16-dehydropregnenolone after intramuscular administration in rats

The pharmacokinetics of 16-dehydropregnenolone (16-DHP), a sterols compound isolated from Solanum lyratum Thunb., was investigated in rats following a Single intramuscular administration (40 mg/kg). The concentration of 16-DHP in rat plasma was determined by a high Performance liquid chromatography (HPLC) method with UV detection. Levonorgestrel was used as the internal Standard (IS). The pharmacokinetic parameters of 16-DHP were derived by non-compartmental method. After a Single intramuscular administration, the maximum plasma concentration (C_max) was (289 ± 25) ng/mL, time to reach C_max(t_max) was (0.38 ± 0.14) h, the elimination half-life (t_1/2) was (2.5 ± 1.1) h, the area under the plasma concentration-time curve from time zero to the time of the last measurable concentration (AUC_(0-t)) was (544 ± 73)ng · h/mL. The results indicated that 16-DHP was absorbed quickly and eliminated rapidly in rats after the intramuscular injection.     

Simultaneous quantitation of atorvastatin and its two active metabolites in human plasma by liquid chromatography/(–) electrospray tandem mass spectrometry

A sensitive, accurate and selective liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the simultaneous quantitation of atorvastatin (AT) and its equipotent hydroxyl metabolites, 2-hydroxy atorvastatin (2-AT) and 4-hydroxy atorvastatin (4-AT), in human plasma. Electrospray ionization (ESI) interface in negative ion mode was selected to improve the selectivity and the sensitivity required for this application. Additionally, a solid phase extraction (SPE) step was performed to reduce any ion-suppression and/or enhancement effects. The separation of all compounds was achieved in less than 6 min using a C₁₈ reverse-phase fused-core^® column and a mobile phase, composed of a mixture of 0.005% formic acid in water:acetonitrile:methanol (35:25:40, v/v/v), in isocratic mode at a flow rate of 0.6 mL/min. The method has lower limit of quantitation (LLOQ) of 0.050 ng/mL for all analytes. The method has shown tremendous reproducibility, with intra- and inter-day precision less than 6.6%, and intra- and inter-day accuracy within ±4.3% of nominal values, for all analytes, and has proved to be highly reliable for the analysis of clinical samples.     

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.     

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.     

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.     

Stability-indicating liquid chromatographic method for the determination of Letrozole in pharmaceutical formulations

A stability-indicating high-performance liquid chromatographic method was developed and validated for the determination of Letrozole in tablet dosage forms. Reversed-phase chromatography was performed on Shimadzu Model LC-Class-Vp with Lichrocart/Lichrosphere 100 C-18 (250 mm×4.6 mm, 5 μm particle size) column with methanol: tetra butyl ammonium hydrogen sulfate (80:20V/V) as mobile phase at a flow rate of 1 mL/min with UV detection at 240 nm. Linearity was observed in the concentration range of 0.5–150 μg/mL (R²=0.9998) with regression equation y=102582x+43185. The limit of quantitation (LOQ) and limit of detection (LOD) were found to be 0.043 and 0.012 μg/mL respectively. The forced degradation studies were performed by using HCl, NaOH, H₂O₂, thermal and UV radiation. Letrozole is more sensitive towards alkaline conditions and very much resistant towards acidic, oxidative and photolytic degradations. The method was validated as per ICH guidelines. The RSD for intra-day (0.78–0.97) and inter-day (0.86–0.96) precision were found to be lesser than 1%. The percentage recovery was in good agreement with the labeled amount in the pharmaceutical formulations and the method is simple, specific, precise and accurate for the determination of Letrozole in pharmaceutical formulations.