High‐Performance Liquid Chromatographic Determination of Parecoxib in Human Plasma and Pharmaceutical Formulations

Abstract

A simple and sensitive RP‐HPLC method for the determination of parecoxib (PXB) in human plasma and pharmaceutical formulations has been developed and validated. The separation of PXB and the internal standard, ibuprofen (IBF) was achieved on a CLC C₁₈ (5 μ, 25 cm×4.6 mm i.d.) column using UV detector at 200 nm. The mobile phase consisted of acetonitrile‐water (92:8 v/v). The linear range of detection was found to be 0.9–18.4 µg/ml (r=0.9985). Intra‐ and inter‐day assay relative standard deviations were observed to be less than 0.3%. The method has been applied successfully for the determination of PXB in spiked human plasma and pharmaceutical preparations. Analytical parameters were calculated and complete statistical evaluation is incorporated.     

Application of High Performance Liquid Chromatography to Separation of Novel Chiral Tetrahedral Heterometal Clusters

A series of novel chiral tetrahedral heterometal clusters have firstly been separated on cellulose tris-(3,5-dimethylphenylcarbamate) stationary phase by high performance liquid chrom-atography, using hexane as the mobile phase with various alcohols as modifiers.     

A Simple and Rapid Reversed Phase High Performance Liquid Chromatographic Method for Quantification of Alprazolam and α-Hydroxyalprazolam in Plasma

Abstract

A simple and rapid reversed-phase liquid chromatographic method for the determination of alprazolam and a-hydroxyalprazolam in plasma is described. Flunictrazepam was used as internal standard. Plasma samples were buffered with sodium borate and extracted with dichloromethane /n-pentane 4:6 v/v for 60 sec on a vortex apparatus. Extraction solvent was evaporated to dryness and extraction residues were reconstituted in the mobile phase. Samples were chromatographed on a 5μ Lichrospher RP-18 column (25cm × 4mm i. d) using acetonitrile/water 40:60 v/v as the mobile phase. The column effluent was monitored at 230nm. The lower limit of detection was 1ng/ml for alprazolam and a-hydroxyalprazolam while the lower limit of quantification was 2ng/ml for both compounds. Peak height and plasma     

Reversed–Phase Liquid Chromatographic Determination of Zaleplon in Human Plasma and its Pharmacokinetic Application

Abstract

A simple, rapid, specific, and reliable high performance liquid chromatographic assay of zaleplon in human plasma has been developed. Reversed‐phase chromatography was conducted using a mobile phase of methanol∶ammonium acetate buffer (50∶50) v/v, pH 3.2 adjusted with orthophosphoric acid, UV detection at 232 nm. After extraction from plasma by precipitation the drug was chromatographed using a C₁₈ reversed‐phase analytical column. The average recoveries of zaleplon from spiked plasma in the concentration range from 0.005–0.2 µg/ml were 93.29%, and their respective CV% was 2.557%. Regression analysis for the calibration plot for plasma standards obtained on three different days for the drug concentrations between 0.005–0.2 µg/ml indicated excellent linearity (r>0.999) and the coefficient of variation of the slopes of the three lines was less than 2%. The limit of detection was 5 ng/ml. Analysis of variance of the data showed no detectable difference in the slopes of the three standard plots (F=3.1, P>0.01). The high correlation coefficients and the similarities in the slopes are good indications of the excellent reproducibility and linearity of the proposed method. The proposed method was applied to study the bioequivalence of a commercial product of zaleplon, using as reference standard the innovator drug product. The study was conducted by using one capsule (1×10 mg) of each of the commercial product and the reference standard in a two‐way open randomized crossover design involving 24 volunteers. The criteria used to assess bioequivalence of the products were AUC (0?∞), C_max, t_max, t_1/2, and K. The obtained values for these parameters were 0.246±0.03 µg h/ml, 0.150±0.013 µg/ml, 1 h, 1.26±0.36 h, and 0.5928±0.1732 h^?1 for product A whereas, for product B they were 0.256±0.044 µgh/ml, 0.142±0.014 µg/ml, 1 h, 1.18±0.33 h, and 0.63±0.1747 h^?1, respectively.     

Development and Validation of High‐Performance Liquid Chromatographic Method for Estimation of Lercanidipine in Rabbit Serum

Abstract

A new, simple, and sensitive reverse‐phase liquid chromatographic method was developed and validated for the estimation of Lercanidipine hydrochloride in rabbit serum using UV detector under isocratic conditions. After subjecting serum to simple and efficient one‐step extraction procedure, 100 µl of sample was injected onto high‐performance liquid chromatography system. The detector response was linear in the concentration range of 25–1000 ng/ml. The developed method was validated as per standard guidelines. Validation demonstrated accuracy, precision, and selectivity of the proposed method. The drug was found to be stable under various processing and storage conditions.     

Quantitative Estimation of Dehydroascorbic Acid and Ascorbic Acid by High-Performance Liquid Chromatography—Application to Human Milk,Plasma, and Leukocytes

Abstract

A ‘high-performance’ liquid chromatographic (HPLC) method for quantitation of dehydroascorbic acid and ascorbic acid and its application to protein-free human milk, blood plasma and leukocytes (buffy layer) is described. In the method, DL-homocysteine was used to convert dehydroascorbic acid quantitatively to ascorbic acid that was measured by reversed phase liquid chromatography. Fresh human milk was found to contain ascorbic acid 54.3±6.5 mg/1 (mean±SEM; n=4) and dehydroascorbic acid 21. 0±9.1 mg/1 (mean±SEM, n=4) when stored at +4°C. The concentration of both forms of ascorbic acid was found to detoriate in similar ratios during storage at +4°C, and pasteurization considerably increased the loss of vitamin C. After pasteurization the milk contained ascorbic acid 8.6±3.4 mg/1 (mean±SEM, n=4) and dehydroascorbic acid 6.6±2.4 mg/1 (mean±SEM, n=4). In plasma the dehydroascorbic acid content (0.16±0.03 mg/1, mean±SEM, n=23) was lower than that of ascorbic acid (9.96±0.75 mg/1, mean±SEM, n=23).

The ascorbic acid concentration in the leukocyte mixtures was 0.21±0.04 mg/10⁹ cells (mean±SEM, n=10) and dehydroascorbic acid concentration 0.09±0.03 mg/10⁹ cells (mean±SEM, n=8). A statistically significant (r=0.599, p<0.05) correlation was established between the concentrations of ascorbic acid in plasma and leukocytes.     

The “On Line” Determination of Cefsulodin by High Performance Liquid Chromatography in CSF and Plasma of Rats

Abstract

After an intraveinous injection of 20 mg.kg^?1 of cefsulodin in anaesthetized rats, the cerebrospinal fluid (CSF) and plasma drug levels were determined on line by HPLC. The CSF was withdrawn from the IIIe venticle at a flow rate of I μl. minute and pushed with a peristaltic pump, via a teflon tube into a “micro vial” inside the WISP vial. At the end of fiveteen minutes, about 15 μ1 of CSF was collected and a small volume (7 μl) was injected by the WISP into the column, the excess of CSF was discarded by another teflon tube inside the “micro-vial”. After the injection, the continous flow of CSF filled the “micro vial”, an injection was running at fiveteen minutes and so on during three hours. Plasma samples were collected every thirty minutes, and after methanol extraction cefsulodin levels were determined by HPLC. Given the low drug levels found in the CSF, this method appeared very sensitive; it was a direct, rapid, automatic technique. Other biological molecules can be determined by this automatic dosage.     

Determination of Vitamin C in Plasma and Dialysate from Uremia Patientsby High Performance Liquid Chromatography with Electrochemical Detection

 Abstract：Aconvenientandvalidmethodforthedeterminationofascorbicacid(AA)anddehydroascorbicacid(DHAA)inplasmaanddialysatefrompatientswithuremiabyhighperformanceliquidchromatographywithelectrochemicaldetectionisdescribedAmixtureof08g/Lmetaphosphoricacidand     

Determination of Cotinine, 3′-Hydroxycotinine,and Their Glucuronides in Urine by Ultra-high Performance Liquid Chromatography

The measurement of the primary nicotine metabolites, cotinine and trans-3′-hydroxycotinine, is a useful biomarker of nicotine exposure and metabolism genetics for smoking cessation research. Herein is described an ultra-high performance liquid chromatography–tandem mass spectrometry method for the determination of these primary nicotine metabolites in urine. Urine samples were diluted one hundred-fold with water and introduced into an ultra-high performance liquid chromatography triple quadrupole mass spectrometer using positive ion electrospray ionization with multiple reaction monitoring. Levels of urinary nicotine metabolites: cotinine, trans-3′-hydroxycotinine, and their respective glucuronides were determined directly using deuterated internal standards and compared with indirect determination by enzymatic hydrolysis. The assay was applied to a community sample of smokers’ urine (n = 280). The assay demonstrated satisfactory performance (relative standard deviation of 1.6–6.5 percent at the 1000 nanograms per milliliter level and >98 percent recovery) suitable for application to smoking studies with a run time less than five minutes. The mean (min-max) levels of cotinine and cotinine-glucuronide were 968 (31-3831) and 976 (9-5607) nanograms per milliliter. The mean (min-max) levels of trans-3′-hydroxycotinine and trans-3′-hydroxycotinine-glucuronide were 3529 (13-21337) and 722 (0-4633) nanograms per milliliter. Direct determination of glucuronide metabolites was superior to indirect measurement using enzymatic hydrolysis, where there was evidence of loss of metabolites during sample preparation. A sensitive and selective ultra-high performance liquid chromatography–tandem mass spectrometry assay was successfully developed for the determination of cotinine, trans-3′-hydroxycotinine, and their glucuronides in urine. The rapid and simple sample preparation makes this assay suitable for high throughput studies involving nicotine metabolism phenotype for both cytochrome P450 2A6 and uridine 5′-diphospho-glucuronosyltransferase, smoking prevalence, and cessation studies.     

Quantitative and Qualitative Analysis of CKD-501, Lobeglitazone, in Human Plasma and Urine Using LC–MS/MS and Its Application to a Pharmacokinetic Study

A simple, rapid and sensitive LC–MS/MS method in positive ion mode was developed and validated to determine CKD-501, lobeglitazone, in human plasma and urine using glipizide as an internal standard (IS). Lobeglitazone is a novel thiazolidinedione (TZDs)-based peroxisome proliferator-activated receptor (PPAR) agonist, used for the management of type-2 diabetes. After mixing the IS, dissolved in acetonitrile, with a plasma or urine sample containing lobeglitazone, 10?μL of supernatant was injected into the LC–MS/MS system. Quantification was performed in the multiple reaction monitoring (MRM) mode using transition of 481.5?→?152.2 (m/z) for lobeglitazone and 446.1?→?321.2 (m/z) for the IS. The method showed good linearity over concentration ranges of 0.5–1,000?ng?mL^?1 for plasma and 0.2–250?ng?mL^?1 for urine (r ²?≥?0.9996). The mean percent extraction recovery of lobeglitazone was 90.8?% for plasma and 87.3?% for urine, while the recoveries of the IS were greater than 86.4?% for both. The intra-day and inter-day precision of plasma ranged from 1.1 to 3.7 and 2.5 to 3.3?% (RSD), respectively, and the intra- and inter-day precision of urine ranged from 1.5 to 2.7 and 3.2 to 3.5?%, respectively. This method is simple, sensitive, and applicable for the pharmacokinetic study of lobeglitazone in human plasma. Most of the urine concentrations of lobeglitazone were below the LLOQ because the lobeglitazone is extensively metabolized.     

Quantitation of Branched Chain α-Keto Acids in Sheep Plasma Using Reversed-Phase High Performance Liquid Chromatography and Quinoxalinol Derivatization

Abstract

The concentration of branched chain α-keto acids in sheep plasma was determined by reversed-phase high performance liquid chromatography using precolumn quinoxalinol formation and ion exchange chromatography for sample preparation. The clear resolution, high degree of precision and accuracy, and relatively simple sample cleanup and derivatization procedures render this technique suitable for the routine analysis of physiological fluids.     

Quantitative and Qualitative Analysis of CKD-501, Lobeglitazone,in Human Plasma and Urine Using LC–MS/MS and Its Application to a Pharmacokinetic Study

A simple, rapid and sensitive LC–MS/MS method in positive ion mode was developed and validated to determine CKD-501, lobeglitazone, in human plasma and urine using glipizide as an internal standard (IS). Lobeglitazone is a novel thiazolidinedione (TZDs)-based peroxisome proliferator-activated receptor (PPAR) agonist, used for the management of type-2 diabetes. After mixing the IS, dissolved in acetonitrile, with a plasma or urine sample containing lobeglitazone, 10 μL of supernatant was injected into the LC–MS/MS system. Quantification was performed in the multiple reaction monitoring (MRM) mode using transition of 481.5 → 152.2 (m/z) for lobeglitazone and 446.1 → 321.2 (m/z) for the IS. The method showed good linearity over concentration ranges of 0.5–1,000 ng mL⁻¹ for plasma and 0.2–250 ng mL⁻¹ for urine (r ² ≥ 0.9996). The mean percent extraction recovery of lobeglitazone was 90.8 % for plasma and 87.3 % for urine, while the recoveries of the IS were greater than 86.4 % for both. The intra-day and inter-day precision of plasma ranged from 1.1 to 3.7 and 2.5 to 3.3 % (RSD), respectively, and the intra- and inter-day precision of urine ranged from 1.5 to 2.7 and 3.2 to 3.5 %, respectively. This method is simple, sensitive, and applicable for the pharmacokinetic study of lobeglitazone in human plasma. Most of the urine concentrations of lobeglitazone were below the LLOQ because the lobeglitazone is extensively metabolized.

     

Separation of the III-α, IX-α, and XIII-α Isomers of Bilirubin and Bilirubin Dimethyl Ester by High Performance Liquid Chromatography

Abstract

Conditions are described for the rapid separation of the III-α, IX-α, and XIII-α isaners of both free bilirubin (BR) and its dimethyl ester (BRDME) by normal-phase HPLC on silica. The HPLC method for BR is an alternative for the tedious TLC method for determination of the isaneric purity of cannercial and other BR samples.     

Improved High Performance Liquid Chromatography of Cyclic Polypeptide Antibiotics—Polymyxins B—and Its Application to Assays of Pharmaceutical Formulations

Abstract

An isocratic high performance liquid chromatographic (HPLC) method for the analysis of polymyxins B1 and B2 is described. The method uses a 25 cm Hypersil—ODS column, a mobile phase containing 22.5% acetonitrile (v/v) in an aqueous phase with tetramethylammonium chloride (TMAC), a flow rate of 1.09 ml/minute and a wavelength of 220 nm for detection. Complete resolution of B1 and B2, and their separation from all other components and/or impurities have been achieved in less than 23 minutes. The capability of the method for the determination of the polymyxin content in pharmaceutical preparations has also been demonstrated.     

Determination of Organotin Compounds in Wine by Microwave-Assisted Extraction and High Performance Liquid Chromatography–Inductively Coupled Plasma Mass Spectrometry

A new analytical procedure for the determination of five organotin compounds in several matrix wine samples is reported. The organotin compounds were extracted by microwave-assisted extraction with n-hexane. Extraction conditions, such as volume of n-hexane required, extraction temperature, and extraction time, were investigated and optimized by an orthogonal array experimental design. The determination of organotin compounds in the final extracts was carried out by liquid chromatography–inductively coupled plasma mass spectrometry. The procedure showed limits of detection between 0.029–0.049 µg · L^?1. The linearity was in the range of 0.5 to 100 µg · L^?1. The precision expressed as relative standard deviation (RSD) was below 9.43%. The developed method was successfully employed to analyze different matrix wine samples, and some analytes were detected at the level of 0.053 to 1.14 µg · L^?1.     

Simultaneous Determination of Flunixin,Phenylbutazone, Oxyphenbutazone and γ-Hydroxyphenylbutazone in Equine Plasma by High-Performance Liquid Chromatography: With Application to Pharmacokinetics

Abstract

A high performance liquid chromatographic method was developed for the simultaneous determination of flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone in equine plasma. Samples of plasma or sera were deproteinated by addition of acetonitrile containing the internal standard naproxen. The concentration step consisted of taking an aliquot of deproteinated plasma, evaporating under nitrogen to dryness and redissolving in mobile phase. The extracts were chromatographed on a Spherisorb 5 μm ODS column using an isocratic mobile phase of methanol (30% v/v), acetonitrile (20% v/v) and pH 3.0 1% acetate buffer (50% v/v) at a flow rate of 1.2 ml/min using naproxen as the internal standard. The detection limit for flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone was 50 ng/ml.

The developed chromatographic method was applied to the determination of equine nonsteroidal anti-inflammatory treatment. Plasma samples from clinically treated horses administered flunixin and phenylbutazone simultaneously are reported. Effect of different anticoagulants used in sampling is reported.     

A Sensitive High Performance Liquid Chromatography Assay for Trospectomycin. An Aminocyclitol Antibiótic,in Human Plasma and Serum

Abstract

This report describes a sensitive, selective and robust assay for the quantification of trospectomycin, an aminocyclitol antibiotic, in human plasma and serum.

This is the first published High Performance Liquid Chramatography (HELC) bioanalytical method for a member of this class of compound.

The method involves the selective solid phase extraction of 6′-n-propyl spectinomycin and the internal standard 6′-n-butyl spectinonycin from 0.5 ml of biofluid, efficient reversed phase high pressure liquid chromatography with post column oxidation, reaction with o-phthaldialdehyde and fluorescence detection. The limit of quantification from 0.5 ml of biofluid is 10 ng/ml.     

Routine Determination of a New 1–4 Dihydropyridine,Oxodipine, in Human Plasma by High Performance Liquid Chromatography with Electrochemical Detection

Abstract

A rapid, specific and reproducible high-performance liquid chromatographic routine assay with electrochemical detection was developed for the determination of Oxodipine in human plasma.

After extraction at alkaline pH by cyclohexane, Oxodipine and its internal standard were chromatographied on a reversed-phase column.

Calibration curves were linear over a concentration range of 1–50 ng/ml with relative errors within-day or between-day not exceeding 8% at any level.

The limit of detection was 30 pg injected based on a signal-to- noise ratio of 7. However, the reliable limit of quantification was 1 ng/ml using 1 ml of human plasma.

A dual-electrode coulometric detector was operated in a screening mode of oxidation, providing a greater specificity and reducing background noise.

This method allowed the complete follow-up of clinical pharmacokinetic studies and drug monitoring in patients.