Simultaneous Determination of Pyridoxine, Meclizine and Buclizine in Dosage Formulations and Human Serum by RP-LC

Rapid liquid chromatographic procedures for analytical quality control of pharmaceutical preparations and human serum containing antihistamine drugs, meclizine and buclizine alone or in combination with pyridoxine are proposed, using acetonitrile:water (80:20) as a mobile phase (pH adjusted to 2.6), methylparaben as internal standard and UV detection was made at 230 nm. The results obtained showed a good agreement with the declared content. The method shows good linearity in the range of 30–10,000 ng mL^?1 for pyridoxine and 25–10,000 ng mL^?1 for meclizine and buclizine serum concentrations with a correlation coefficient 0.9999 (inter- and intra-day CV < 3.91%). The recovery was >97.8%. The proposed method may be used for the quantitative analysis of meclizine and buclizine alone or in combination with pyridoxine from raw materials, in bulk drugs, dosage formulations and in serum.     

Simultaneous Determination of Tranexamic Acid and Losartan Potassium in Dosage Formulations and Human Serum by RP-LC

Rapid liquid chromatographic procedure for analytical quality control of pharmaceutical preparations and human serum containing drugs, tranexamic acid together with losartan potassium are proposed, using acetonitrile: water (50:50), adjusting pH to 2.6 with phosphoric acid as a mobile phase, UV detection at 205 nm and propylparaben sodium was used as internal standard. The results obtained showed a good agreement with the declared contents. The method shows good linearity in the range of 40–10,000 ng mL^?1 for tranexamic acid serum concentrations with a correlation coefficient 0.9999 (inter- and intra-day CV <3.18) and in the range 5–10,000 ng mL^?1 for losartan potassium serum concentrations with a correlation coefficient 0.9999 (inter- and intra-day CV <3.61). The recovery was >97.8%. The proposed method may be used for the quantitative analysis of tranexamic acid and losartan potassium alone or in combination from raw materials, in bulk drugs, dosage formulations and in serum.     

Simultaneous Determination of Abacavir,Efavirenz and Valganciclovir in Human Serum Samples by Isocratic HPLC-DAD Detection

A rapid, sensitive, and specific reverse phase high performance liquid chromatography with diode array detection procedure for the simultaneous determination of abacavir, efavirenz and valganciclovir in spiked human serum is described. Separation was performed on a 5 μm Waters Spherisorb column (250 × 4.6 mm ID) with acetonitrile: methanol:KH₂PO₄ (at pH 5.00) (40:20:40 v/v/v) isocratic elution at a flow rate of 1.0 mL min⁻¹. Calibration curves were constructed in the range of 50–30,000 ng mL⁻¹ for abacavir and efavirenz, and 10–30,000 ngmL⁻¹ for valganciclovir in serum samples. The limit of detection and limit of quantification concentrations of the HPLC method were 3.80 and 12.68 ng mL⁻¹ for abacavir, 2.61 and 8.69 ng mL⁻¹ for efavirenz, 1.30 and 4.32 ng mL⁻¹ for valganciclovir. The method has been applied, without any interference from excipients or endogenous substances, for the simultaneous determination of these three compounds in human serum.

     

GC Determination of Famotidine,Ranitidine, Cimetidine,and Metformin in Pharmaceutical Preparations and Serum Using Methylglyoxal as Derivatizing Reagent

A capillary gas chromatography (GC) procedure has been developed for the determination of four pharmaceutical preparations (famotidine, ranitidine, cimetidine, and metformin) after precolumn derivatization with methylglyoxal (MGo). GC was carried out using an HP-5 column (30 m × 0.32 mm id) at an initial column temperature of 90 °C for 2 min, followed by heating rate of 25 °C min⁻¹ up to 265 °C. Nitrogen flow rate was 2.5 mL min⁻¹ with split ratio 10:1. A linear calibration curve was obtained within 50–1,000 ng mL⁻¹ and the limit of detection (LOD) was within 17–25 ng mL⁻¹. The derivatization, GC elution, and separation were repeatable in terms of retention time and peak height/peak area with relative standard deviation within ±4.6 %. The procedure was applied to the determination of the drugs in pharmaceutical preparations and the sera of volunteers who were given oral doses of the drugs. The results of the analysis agreed with the labeled values of the pharmaceutical preparations and were 147–4,903 ng mL⁻¹ in serum with an RSD within 1.0–4.2 %, after ingestion of a single dose of 40–500 mg of active ingredient in a tablet.

     

HPLC and UPLC Methods for the Simultaneous Determination of Enalapril and Hydrochlorothiazide in Pharmaceutical Dosage Forms

High efficiency and less elution are the basic requirements of high-speed chromatographic separation. In this study, a new gradient reverse phase chromatographic methods were developed using HPLC and UPLC systems for simultaneous determination of enalapril maleate (ENL) and hydrochlorothiazide (HCZ) in pharmaceutical dosage forms. The chromatographic separations of ENL and HCZ were achieved on a Waters μ-Bondapak C 18, (300 × 3.9 mm, 10 μm) and Waters Acquity BEH C18 (100 × 2.1 mm, 1.7 μm) columns for HPLC within 5.30 min and UPLC within a short retention time of 1.95 min, respectively. A linear response was observed over the concentration range 0.270–399 μg mL⁻¹ of ENL, 0.260–399 μg mL⁻¹ of HCZ for HPLC system and 0.270–399 μg mL⁻¹ of ENL and 0.065–249 μg mL⁻¹ of HCZ for UPLC system. Also, limit of detection for ENL was 1.848 ng mL⁻¹ and 31.477 ng mL⁻¹ for HCZ, 2.804 ng mL⁻¹ for ENL and 2.943 ng mL⁻¹ for HCZ using HPLC and UPLC, respectively. The proposed methods were validated according to ICH guideline with respect to precision, accuracy, and linearity. Forced degradation studies were also performed for both compounds in bulk drug samples to demonstrate the specificity and stability indicating power of the HPLC method. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency, and resolution.

     

Development and Validation of an Stability Indicating RP-LC Method for Determination of Imatinib Mesylate

A new, sensitive and stability indicating liquid chromatographic method has been developed for the determination of imatinib mesylate (IM). Efficient chromatographic separation was achieved using a C₁₈ column with simple mobile phase combination delivered in an isocratic mode and quantitation was carried out using ultraviolet detection. For the first time, a novel microwave assisted degradation procedure was employed for stress testing studies. In addition, orthogonal separation technique was applied to demonstrate selectivity of the proposed method. The method has demonstrated excellent linearity over the range of 25–1,600 ng mL⁻¹. Moreover, the method was found to be sensitive with a low limit of detection (3.35 ng mL⁻¹) and limit of quantitation (10.16 ng mL⁻¹). The method has shown good and consistent recoveries (99.35–100.69%) with low intra- and inter-day relative standard deviation (RSD) (<2.5%). Experimental design confirmed that peak area was unaffected by small changes in critical factors, in robustness study. The validated method was successfully applied for determination of IM in pharmaceutical formulations.

     

Development and Validation of LC-UV for Simultaneous Estimation of Rosiglitazone and Glimepride in Human Plasma

A simple, sensitive and specific liquid chromatographic method with UV detection (228 nm) was developed for the simultaneous estimation of rosiglitazone and glimepride in human plasma. Rosiglitazone and glimepride were extracted from plasma using liquid–liquid extraction. Separation was achieved with an RP C₁₈ Column using a mixture of phosphate buffer (50 mM) with octane sulfonic acid (10 mM), methanol and acetonitrile as a mobile phase (55:10:35, v/v). pH was adjusted to 7.0. Amlodipine was used as an internal standard (IS). LOD of the method was found to be 20 ng mL⁻¹ for both drugs. Results were linear over the studied range 40.994–2007.556 ng mL⁻¹ for rosiglitazone (r ² ≥ 0.99) and 41.066–2094.84 ng mL⁻¹ for glimepride( r ² ≥ 0.99). The method was found to be simple, selective, precise and reproducible for the estimation of both drugs from spiked human plasma.

     

Simultaneous LC–MS Analysis and of Wogonin and Oroxylin A in Rat Plasma,and Pharmacokinetic Studies After Administration of the Active Fraction from Xiao-Xu-Ming Decoction

A rapid and specific high-performance liquid chromatographic method coupled with electrospray ionization mass spectrometric detection has been developed and validated for identification and quantification of wogonin and oroxylin A in rat plasma. Wogonin, oroxylin A, and diazepam (internal standard) were extracted from plasma samples by liquid–liquid extraction with ethyl acetate. Chromatographic separation was achieved on a C₁₈ column with acetonitrile–0.6% aqueous formic acid 35:65 (v/v) as mobile phase at a flow rate of 0.2 mL min⁻¹. Detection was performed with a single-quadrupole mass spectrometer in selected-ion-monitoring (SIM) mode. Linearity was good within the concentration range 14.4–360 ng mL⁻¹ for wogonin and 10.8–271 ng mL⁻¹ for oroxylin A; the correlation coefficients (r ²) were 0.9999. The intra-day and inter-day precision, as RSD, was below 12.4%, and accuracy ranged from 81.1 to 111.9%. The lower limit of quantification was 14.4 ng mL⁻¹ for wogonin and 10.8 ng mL⁻¹ for oroxylin A. This method was successfully used in the first pharmacokinetic study of wogonin and oroxylin A in rat plasma after oral administration of the active fraction from Xiao-xu-ming decoction.

     

Determination of Three Nonsteroidal Anti-Inflammatory Drugs in Human Plasma by LC Coupled with Chemiluminescence Detection

A simple and sensitive method was developed for the determination of three nonsteroidal anti-inflammatory drugs (NSAIDs)—ibuprofen, naproxen and fenbufen in human plasma. The method involved in column liquid chromatographic separation and chemilumenescence (CL) detection based on the CL reaction of NSAIDs, potassium permanganate (KMnO₄) and sodium sulfite (Na₂SO₃) in sulfuric acid (H₂SO₄) medium. The chromatographic separation was carried out using a reversed-phase C₁₈ column, which allowed the selective determination of the three medicines in the complicated samples. The special features of the CL detector provided lower LOD for determination than that of existing chromatographic alternatives. The results indicated that the linear ranges were 0.01–10.0 μg mL⁻¹ for ibuprofen, 0.001–1.0 μg mL⁻¹ for naproxen, and 0.01–10.0 μg mL⁻¹ for fenbufen. The limits of detection were 0.5 ng mL⁻¹ for ibuprofen, 0.05 ng mL⁻¹ for naproxen and 0.5 ng mL⁻¹ for fenbufen (S/N = 3). All average recoveries were in the range of 90.0–102.3%. Finally, the method had been satisfactorily applied for the determination of ibuprofen, naproxen and fenbufen in human plasma samples.

     

Determination of Anilines and Toluidines in Water by Salt-Assisted Dispersive Liquid–Liquid Microextraction Combined with GC-FID

Dispersive liquid–liquid microextraction (DLLME) assisted with salting-out was applied for the determination of five aromatic amines in water samples by using gas chromatography with flame ionization detection. In this extraction method, several factors influencing the extraction efficiency of the target analytes, such as extraction and disperser solvent type and their volume, salt addition and amount, and pH, were studied and optimized. Under the optimal DLLME conditions, good linearity was observed in the range of 4–1,000 ng mL⁻¹ with the RSDs from 1.2 to 7.9 %. The LODs based on S/N of 3 ranged from 0.2 to 3.4 ng mL⁻¹ and the enrichment factors ranged from 207 to 4,315. The proposed method was successfully applied to the water samples collected from the tap and the lake, and the relative recoveries were in the range of 87.7–108.4 %.

     

Development and Validation of an RP-HPLC Method for Determination of Valganciclovir in Human Serum and Tablets

This paper describes the validation of an isocratic high-performance liquid chromatographic method for the assay of valganciclovir in raw materials, tablets and human serum samples. Valganciclovir and fluvastatin (internal standard) were well separated using a reversed phase column and a mobile phase consisting of a mixture of acetonitrile:methanol:KH₂PO₄ (0.02 M) (40:20:40; v/v/v) (at pH 5.0). The mobile phase was pumped at 1.0 mL min⁻¹ flow rate and valganciclovir was detected by diode-array detection at 255 nm. The retention times for valganciclovir and fluvastatin were 3.41 and 5.60 min, respectively. A linear response (r > 0.999) was observed in the range of 10–30,000 ng mL⁻¹ in mobile phase and serum. The limit of detection and limit of quantification were found as 2.95 and 9.82 ng mL⁻¹ in mobile phase and 1.73 and 5.77 ng mL⁻¹ in human serum samples, respectively. Validation parameters as precision, accuracy, selectivity, reproducibility and system suitability tests were also determined. The method can be used for valganciclovir assay of tablets and human serum samples as the method separates valganciclovir from tablet excipients and endogenous substances.

     

LC–MS Simultaneous Determination of Itopride Hydrochloride and Domperidone in Human Plasma

A rapid, simple, sensitive and specific liquid chromatography–tandem mass spectrometry method was developed and validated for simultaneous quantification of itopride hydrochloride and domperidone in human plasma. Both drugs were extracted by liquid–liquid extraction with ethyl acetate and saturated borax solution. The chromatographic separation was performed on a reversed-phase C18 column with a mobile phase of water–methanol (2:98, v/v) containing 0.5% formic acid. The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The assay exhibited linearity over the concentration range of 3.33–500 ng mL⁻¹ for itopride hydrochloride and 3.33–100 ng mL⁻¹ for domperidone in human plasma. The precursor to product ion transitions of m/z 359.1–72.3 and 426.0–147.2 were used to measure itopride hydrochloride and domperidone respectively. The method was found suitable for the analysis of plasma samples collected during phase 1 pharmacokinetics study of itopride HCl 50 mg and domperidone 20 mg in 12 healthy volunteers after single oral doses of the combination drug.

     

RP-LC Determination and Pharmacokinetic Study of Ferulic Acid and Isoferulic Acid in Rat Plasma After Taking Traditional Chinese Medicinal-Preparation: Guanxinning Lyophilizer

A sensitive, simple, and accurate method for determination and pharmacokinetic study of ferulic acid and isoferulic acid in rat plasma was developed using a reversed-phase column liquid chromatographic (RP-LC) method with UV detection. Sample preparations were carried out by protein precipitation with the addition of methanol, followed by evaporation to dryness. The resultant residue was then reconstituted in mobile phase and injected into a Kromasil C₁₈ column (250 × 4.6 mm i.d. with 5 μm particle size). The mobile phase was methanol-1% formic acid (33:67, v/v). The calibration plots were linear over the range 5.780–5780 ng·mL⁻¹ for ferulic acid and 1.740–348.0 ng·mL⁻¹ for isoferulic acid. Mean recoveries were 85.1% and 91.1%, respectively. The relative standard deviations (RSDs) of within-day and between-day precision were not above 15% for both of the analytes. The limits of quantification were 5.780 ng·mL⁻¹ for ferulic acid and 1.740 ng·mL⁻¹ for isoferulic acid. This RP-LC method was used successfully in pharmacokinetic studies of ferulic acid and isoferulic acid in rat plasma after intravenous injection of Guanxinning Lyophilizer.

     

TLC Determination of Amitriptyline HCl,Trifluoperazine HCl,Risperidone and Alprazolam in Pharmaceutical Products

A simple, sensitive, and precise thin layer chromatographic (TLC) method for simultaneous analysis of psychopharmacological drugs like amitriptyline HCl, trifluoperazine HCl, risperidone and alprazolam in their single dosage forms has been developed, validated, and used for determination of the compounds in commercial pharmaceutical products. The TLC separation was carried out on Merck TLC aluminium sheets of silica gel 60 F254 using carbon tetrachloride:acetone:triethylamine (8:2:0.3, v/v/v), as mobile phase. Densitometric measurements of their spots were achieved at 250 nm over the concentration range for amitriptyline HCl (50–1,200 ng spot⁻¹), trifluoperazine HCl (50–1,200 ng spot⁻¹), risperidone (100–2,400 ng spot⁻¹) and alprazolam (25–600 ng spot⁻¹). Limit of detection (LOD) for amitriptyline HCl (20 ng spot⁻¹), trifluoperazine HCl (20 ng spot⁻¹), risperidone (40 ng spot⁻¹) and alprazolam (5 ng spot⁻¹) was obtained. The study showed that TLC was sensitive and selective for determination of amitriptyline HCl, trifluoperazine HCl, risperidone and alprazolam using a single mobile phase. This proposed method is able for simultaneous determination of psychopharmacological drugs and also applicable for analysis of pharmaceutical formulations.

     

Accuracy Profile Theory for the Validation of an LC–MS-MS Method for the Determination of Risperidone and 9-Hydroxyrisperidone in Human Plasma

A sensitive and specific LC–MS-MS method is described for the simultaneous quantification of risperidone and 9-hydroxyrisperidone in human plasma. After extraction with tert-butyl methyl ether, plasma samples were separated on an Atlantis HILIC Silica C18 column (4.6 × 150 mm, 5 μm)with a mobile phase of ammonium formate buffer (10 mM, pH 4.0)/acetonitrile (40/60, v/v). Detection was by MS-MS. The method was fully validated according to the accuracy profile theory. It is based on β-expectation tolerance interval for the total measurement error which includes trueness and intermediate precision. The measurement uncertainty derived from β-expectation tolerance interval was estimated at each of the validation standards. The linearity fitted well over the range of 0.11–26.75 ng mL⁻¹ for risperidone with an LLOQ of 0.11 ng mL⁻¹, and for 9-hydroxyrisperidone, at a range of 0.15–37.8 ng mL⁻¹ with an LLOQ of 0.15 ng mL⁻¹. The intra- and inter-batch precision of risperidone were <5.71 and 8.22%, respectively. For 9-hydroxyrisperidone, the data were 5.78 and 6.48%. The recoveries were 88.78% (risperidone) and 70.35% (9-hydroxyrisperidone). The developed method was applied to a pharmacokinetic study of risperidone.

     

Analysis of Bile Acids Profile in Human Serum by Ultrafiltration Clean-up and LC-MS/MS

Bile acids (BAs) are useful biomarkers for the diagnosis of many diseases. The pathologies related to bile acid synthesis are often expressed in the first years of life and may lead to serious liver injury. Here we present a sensitive and rapid method for the analysis of the main 14 bile acids in human serum by liquid chromatography-tandem mass spectrometry. The chromatographic separation is performed using a core–shell column which provides improved separation, highly desirable considering the small structural differences among the analytes. All isomeric BAs of interest were resolved in less than 9 min. Sample pretreatment consisted in ultrafiltration of serum after addition of methanol by means of centrifugal filter devices. The calculated LOQs ranged between 2 and 5 ng mL⁻¹ with linearity of the calibration curves in the 5–5,000 ng mL⁻¹ range for all the BAs. The extraction recoveries for all the analytes were higher than 80 %. Intra-day and inter-day coefficients of variation were all below 15 %. The method proposed has been validated and has been applied for the analysis of serum of pediatric patients. This simple procedure allowed minimal consumption of serum sample (about 100 μL) and a rapid assay, easily implementable in routine analysis.

     

Development and Validation of an HPLC–UV–Vis Method for the Determination of Proparacaine in Human Aqueous Humour

A sensitive high performance liquid chromatographic method has been developed and validated for the determination of proparacaine in human aqueous humour. The procedure involved extraction of proparacaine from aqueous humour with cyclohexane. The separation was achieved using a Bondesil C₈ (250 × 4.6 mm i.d., particle size 5 μm) analytical column with a mobile phase consisted of acetonitrile and sodium dihydrogen phosphate (pH 3.0, 20 mM) (30:70, v/v). Proparacaine and lidocaine (internal standard, IS) detection was performed by UV–Vis detector at 220 nm. The retention times for proparacaine and IS were 12.01 and 5.58 min, respectively. HPLC–UV–Vis method was linear in the range of 75–4,000 ng mL⁻¹. The limit of detection (LOD) was 25 ng mL⁻¹ and the limit of quantification (LOQ) of proparacaine was found to be 75 ng mL⁻¹ (RSD ≤ 15%, n = 6). In intra- and inter-day precision and accuracy analysis, the relative standard deviation was found to be in the range of 0.96 and 7.98%, the bias values were 0.64 and 3.33%. Recovery of proparacaine from human aqueous humour was 99.98% at 500 ng mL⁻¹. Proparacaine solutions were stable at least 6 months at +4 and −20 °C. Proparacaine levels of aqueous humour in fifteen volunteers’ were in the range of 80.21 and 459.00 ng mL⁻¹. According to system suitability tests and Shewhart’s quality control charts the proparacaine responses were in the acceptance ranges. Developed method was providing a sufficient quality at least over 3 months for determination of proparacaine in human aqueous humour.

     

Hollow-Fiber-Based LPME as a Reliable Sampling Method for Gas-Chromatographic Determination of Pharmacokinetic Parameters of Valproic Acid in Rat Plasma

A sensitive and simple method based on two-phase liquid-phase microextraction in porous hollow fiber followed by gas chromatography-flame ionization detection was developed for quantification and pharmacokinetic study of valproic acid (VPA, an antiepileptic drug) in rat plasma after oral administration of pure sodium valproate (25 mg kg⁻¹). Some parameters such as type of organic solvent, pH of sample solution, stirring speed, salt addition, extraction time, and volume of sample that affected extraction efficiency of VPA were optimized. Under optimized microextraction conditions, VPA was extracted with 10 μL 1-octanol from 0.5 mL rat plasma previously diluted with 4.5 mL acidified and salinated water (pH 2) using 1-octanoic acid as internal standard. The limit of detection was 17 ng mL⁻¹ with linear response over the concentration range of 50–10,000 ng mL⁻¹ with correlation coefficient higher than 0.998. The developed method was successfully applied to determination of pharmacokinetic parameters such as t _max (peak time in concentration–time profile), C _max (peak concentration in concentration–time profile), t _1/2 (elimination half-life), AUC_0–t (area under the curve for concentration versus time), clearance, and apparent distribution volume in rats following oral administration of VPA.

     

Determination of DNA Methylation and Hydroxymethylation Levels in Biological Samples by Field-Amplified Sample Injection-Capillary Zone Electrophoresis with UV Detection

In this study, a method of field-amplified sample injection coupled with capillary zone electrophoresis with ultraviolet detection was established for evaluation of DNA methylation and hydroxymethylation levels in biological materials. By modifying an existing method, the separation of cytosine (C), 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) was performed on an uncoated capillary column (40 cm × 75 μm I.D.) using 300 mmol L⁻¹ tris solution (pH 2.90) as running buffer and detected at 280 nm. The detection limits (S/N = 3) of the method were 0.004 ng mL⁻¹ for cytosine (C), 0.01 ng mL⁻¹ for 5-methylcytosine (5-mC), and 0.02 ng mL⁻¹ for 5-hydroxymethylcytosine (5-hmC). The proposed method has been successfully applied to the evaluation of DNA methylation and hydroxymethylation levels of blood samples from 15 hepatocellular carcinoma patients and 5 liver cirrhosis patients and liver tissues from 50 pairs of tumor and matched tumor-adjacent samples.

     

Simultaneous Determination of Pregabalin, Sildenafil and Its Active Metabolite in Rat Plasma Utilising SPE Followed by LC�CMS�CMS

A sensitive and selective analytical method for the quantification of pregabalin, sildenafil and the active desmethyl metabolite of sildenafil (UK-103320) has been developed. The method can simultaneously quantify the three analytes within the expected in vivo concentration ranges using 50 ??L of rat plasma. It utilises solid-phase extraction followed by high performance liquid chromatography coupled with tandem mass spectrometry. Quantitation in rat plasma demonstrated good accuracy and precision over the following dynamic ranges for each analyte: pregabalin (70?C10,000 ng mL^?1), sildenafil (1?C2,000 ng mL^?1) and UK-103320 (1?C2,000 ng mL^?1). For each analyte, the following lower limits of quantitation were obtained: 70 ng mL^?1 for pregabalin and 1 ng mL^?1 for sildenafil and UK-103320, respectively. The method was successfully used to analyse plasma samples from rats when pregabalin and sildenafil were administered in combination.