A Simple Isocratic High-Performance Liquid Chromatographic (HPLC) Determination of Naproxen in Human Plasma using a Microbore Column Technique

Abstract

A simple and sensitive HPLC method was developed for the determination of naproxen in human plasma. The assay employs a microbore column packed with a C18 reversed-phase material (5 μm ODS Hypersil) with an isocratic mixture of acetonitrile and 10 mM phosphate buffer, pH 2.5 (40:60, v/v) as the mobile phase. The mobile phase was pumped at a flow rate of 0.5 ml/min. For sample analysis 200 μl of acetonitrile containing internal standard (flurbiprofen) was added to 100 μl of plasma. After centrifugation 10 mM phosphate buffer, pH 7.4 (200 μl) was added to the tube, then vortexed and centrifuged. The supernatant (20 μl) was injected onto the HPLC column. The chromatographic separation was monitored by a fluorescence detector at an emission wavelength of 350 nm with an excitation wavelength of 225 nm. The direct precipitation of plasma protein using acetonitrile gave a good recovery for both naproxen and the internal standard. The detection limit was 0.1 μg/ml for naproxen. The intra- and inter-assay coefficients of variation at different concentrations evaluated were less than 10%.     

Rapid Assay for the Determination of Tolfenamic Acid in Pharmaceutical Preparations and Biological Fluids by High-Performance Liquid Chromatography

Abstract

An improved reversed-phase High-Performance Liquid Chromatographic (HPLC) method using UV detection, at 282 nm, is described for the determination of tolfenamic acid in the presence of caffeine, as internal standard, in pharmaceutical preparations and biological fluids. Sample analyses are performed with a Lichrosorb-RP18, 10 μm, 250×4 mmLD., column using acetate buffer, (pH 4.6 and constant ionic strength 0.05 M) methanol (18:82) as eluent, at a flow rate of 1.9 ml/min. The retention time is 1.44 min for caffeine and 2.62 min for tolfenamic acid. The absolute detection limit is 0.5 ng in the presence and 0.9 ng in the absence of internal standard and linearity is observed up to 100 ng injected. The method involves the use of solid     

An HPLC Method for the Determination of Verapamil and Norverapamil in Human Plasma

Abstract

A high performance liquid chromatographic method is presented for the determination of verapamil and its metabolite norverapamil in human plasma. Verapamil and norverapamil are extracted from plasma basified with 0.5M dibasic sodium phosphate (pH 9.5) using ethyl acetate containing trimipramine as an internal standard. A reverse-phase cyanopropylsilane column was used with a mobile phase of 65% acetonitrile and 35% 0.02M acetate buffer (pH 7.0). The minimum detectable limit was 2 ng/ml of plasma. The effect of the pH, molarity, and percent acetonitrile of the mobile phase on the capacity factor was studied. Possible interferences from other drugs administered concurrently are presented.     

Determination of asymmetric and symmetric dimethylarginines in human plasma by HPLC with electrochemical detection

A new HPLC method was developed and validated for the determination of asymmetric and symmetric dimethylarginines and l ‐arginine in human plasma. After SPE and evaporation of the eluate, the samples were derivatised with an o‐phthaldialdehyde reagent containing 3‐mercaptopropionic acid. The derivatives formed were analysed by isocratic RP‐HPLC with electrochemical detection at +320 mV. The mobile phase consisted of 50 mM phosphate buffer (pH 6.1) containing 10% v/v acetonitrile, the flow rate was 1 mL/min. The retention times of all compounds including monomethylarginine (internal standard) were <24 min. The LODs (S/N 3:1) were 0.012 μM for both dimethylarginines and 0.013 μM for l ‐arginine; the linearity of the method was from 0.1 to 20 μM for both dimethylarginines and from 1 to 200 μM for l ‐arginine. Absolute extraction recoveries measured for all analytes ranged from 85 to 88%.     

Analysis of Plasma Diethyldithiocarbamate–A Metabolite of Disulfiram

Abstract

A reversed-phase high-performance liquid chromatographic analysis was developed for diethyldithiocarbamate in plasma. Following treatment of plasma (1 ml) with methyl iodide (250 μl), biphenyl (internal standard, 1.8 μg) was added in chloroform (6 ml). After shaking (30 min.), the chloroform was separated and evaporated under nitrogen (to 50 μl). Acetonitrile (250 μl) was added and the solution was again evaporated under nitrogen (to 100 μl). Aliquots (25 μl) were chromatographed using acetonitrile: acetate buffer (65:35, pH 4) at 2.5 ml/min on a 5 micron C-8 column with detection at 276 nm. Recovery of methyldiethyldithiocarbamate (MeDDC) was 92.5 ± 3.2%. Retention times and theoretical plates for MeDDC and biphenyl were 3.0 and 4.6 min., and 4660 and 6336 respectively. The analysis was linear over the range 25 to 400 ng/ml with a coefficient of variation of 3.2%. Analysis of samples after intravenous disulfiram (10 mg) administration to rats yielded a total body clearance of 343 ml/min. This supports the view that metabolism is principally by extra-hepatic routes.     

Analysis of tofisopam in human serum by column-switching semi-micro high-performance liquid chromatography and evaluation of tofisopam bioequivalency

A rapid and sensitive column-switching semi-micro HPLC method is described for the direct analysis of tofisopam in human serum. The sample (100 microL) was directly injected onto the precolumn (Capcell Pak MF Ph-1), where unretained proteins were eluted to waste. Tofisopam was then eluted into an enrichment column using 13% acetonitrile in 50 mM phosphate buffer (pH 7.0) containing 5 mM sodium octanesulfonate and subsequently into the analytical column using 43% acetonitrile in 0.1% phosphoric acid containing 5 mM sodium octanesulfonate. The detection limit (2 ng/mL), good precision (CV < or = 4.2%) and speed (total analysis time 24 min) of the present method were sufficient for drug monitoring. This method was successfully applied to a bioequivalence test of two commercial tofisopam tablets.     

Sensitive Determination of Piritramide in Human Plasma by High-Performance Liquid Chromatography

Abstract

A selective and sensitive method for the determination of piritramide in human plasma is described. After addition of 50 μl of 2 M ammonia and 20 μl of aqueous promethazine solution (100 ng/10 μ1) as an internal standard, 1 ml of plasma was extracted with 5 ml of toluene (extraction efficiency: 93.9 × 2.6%; mean × S. D.; n = 5). HPLC was performed with a phenyl hypersil NC-04 column, particle size 5 μm, 250 × 4 mm I. D.; mobile phase: 8 parts of acetonitrile and 2 parts of 10 mM potassium phosphate buffer (pH 3. 3). The flow rate was set to 2 ml/min and the column temperature was 22°C. The assay was linear in a concentration range of 3.75 ? 3000 ng/ml (r = 0.999), with a lower limit of detection of 3 ng/ml. The precision was determined using spiked plasma samples (15 ng/ml; 300 ng/ml), with coefficients of variation of 6.1 and 5.9% (intraday; n = 5) and 6.5 and 0.2% (interday; n = 3). In the range of 5.6 ? 1500 ng/ml, the accuracy of the assay was 2.82%. The method was used for the determination of piritramide plasma concentrations in patients receiving intra- or postoperative analgesia.     

High-Performance Liquid Chromatographic Determination of Atenolol in Tablets

Abstract

A reversed phase high-performance liquid chromatographic method was developed for the determination of atenolol in four oral 100 mg atenolol preparations.

An aliquot of the sample is dissolved in a mobile phase consisting of 0.0612 M potassium hydrogen phosphate - isopropanol-tetrahydrofuran (84:10:6) v/v). The pH was adjusted to 6.7 with phosphate buffer. Nicotinamide was used as internal standard and chromatographed on a Pinkerton column ISRP (GFF-S5–80) 5 μm, 150 × 4.6 mm i.d. The applied column is convenient for the assay at least 90 samples of atenolol without degrading column performance. The detection was performed at 272 nm. The retention time for atenolol was 5.07 min.

The proposed HPLC method was found to be suitable for the rapid and precise routine analysis of atenolol in tablets.     

基于集成化多柱二维液相色谱系统分析血清中的氨磺必利

快速准确的治疗药物监测对于临床上确保患者用药有效性及安全性至关重要,同时也能够确定患者用药依从性,制定个性化给药方案。该文以两支疏水性略有差异的反相分离柱Supersil ODS2和SinoChrom ODS-BP,及强阳离子交换捕集柱Supersil SCX构建了基于集成化的多柱二维液相色谱系统。通过二维色谱接口,以pH 3.0的磷酸缓冲液调整第一维分离后的洗脱液组成,降低有机相含量并维持pH,改善了中心切割模式下样品转移和捕集的效率。利用该多柱二维液相色谱系统发展了血清中氨磺必利的二维液相色谱检测方法,血清样品经过高氯酸和甲醇混合液沉淀蛋白质并离心后直接300 μL大体积进样,以乙腈/磷酸缓冲液(25 mmol/L, pH 3.0)(20/80, v/v)作为第一维分离流动相,磷酸缓冲液(25 mmol/L, pH 3.0)作为捕集过程的稀释流动相,乙腈/磷酸缓冲液(25 mmol/L, pH 7.0)(25/75, v/v)作为第二维分离流动相,12 min内即可完成分析。方法在10~200 ng/mL的范围内线性相关性良好(r=0.9998)。样品在50 ng/mL和100 ng/mL两个加标浓度下的回收率稳定,在73.7%~76.8%之间。方法的检出限为7.28 ng/mL,定量限为24.27 ng/mL,能够满足《神经精神药理学治疗药物检测共识指南》中推荐的药物监控范围要求。由于该系统日常使用及维护成本较低,且能够实现自动化分析,故该方法适合在临床上用于治疗药物监测研究。     

Direct analysis of clomipramine in human plasma by microbore high performance liquid chromatography with column-switching

Summary An automated microbore, liquid chromatographic method with column-switching was developed for the determination of clomipramine from human plasma samples. After direct injection of samples (60 μL), plasma proteins and clomipramine were separated in size-exclusion mode using 20% acetonitrile in 20 mM phosphate buffer (pH 7.0) on Capcell Pak MF Ph-1 precolumn (10×4 mm I.D.). By valve switching, a fraction containing clomipramine was directed to an intermediate column for subsequent main separation on a microbore C₁₈ column (250×1.5 mm I.D.) using 50% acetonitrile in 20 mM phosphate buffer (pH 2.5) at 0.1 mL min⁻¹. The method was advantageous for rapidity (total analysis time: 15 min), reproducibility (C.V.<4.8%), and increased sensitivity (1 ng mL⁻¹). The linearity of response was good (r ²≥0.999) over the concentration range 1–250 ng mL⁻¹.     

Simultaneous determination of free and bound adriamycin, adriamycinol, adriamycinone and duanorubicin in plasma using column-switching technique and protein-coated pre-columns

Adriamycin, adriamycinol, adriamycinone and duanorubicin were simultaneously determined by the development of an on-line plasma clean-up system. A short protein-coated Lichrosorb, RP-8, RP-2, CN and muBondapak phenyl as well as ODS silica have been examined for their performance as pre-columns. The drugs and metabolites were separated from weakly retained plasma components through two steps; phosphate buffer saline, pH 7.4 and 15% acetonitrile in 0.1 M sodium dihydrogen phosphate, pH 3. The chromatographic conditions were: ODS/TM column, flow rate 1 ml/min, 35% acctonitrile in 0.1 M sodium dihydrogen phosphate (pH 3) containing 0.3% heptafluorobutyric acid as mobile phase. The detection was carried out using fluorescence monitor operated at an emission 555 nm and excitation 460 nm. Good resolution was obtained within 13 min. This method is reproducible for analysis of drugs and metabolites (99.3-100.1%, CV < 2%) in plasma.     

A Reversed-Phase HPLC Method For Determining Camptothecin In Plasma With Specificity For the Intact Lactone Form of the Drug

Abstract

Camptothecin is a pentacyclic indole alkaloid with a terminal α-hydroxy-δ-lactone ring, which in aqueous media at physiological pH, exists in equilibrium with the dissociated open-lactone carboxylate. the rate of equilibration between the two components is slow enough to permit their separation by reversed-phase HPLC. Selective determination of the intact lactone form of the drug was achieved by direct analysis of plasma samples immediately upon deproteinization with a solution of the internal standard in methanol chilled to ?70°C. Acidification of the sample to pH 2 with perchloric acid prior to protein precipitation effected complete lactonization of the carboxylate and, therefore, provided a measure of total drug levels. Plasma concentrations of the carboxylate may be calculated from the difference between total drug and intact lactone determinations. Chromatography was performed on a 5 μm Ultrasphere ODS column (4.6 mm × 25 cm) preceded by a 1.5 cm RP-18 Brownlee Guard column with an eluent composed of acetonitrile-0.1 M ammonium acetate buffer, pH 5.5 (28:72, v/v) with 1 mM sodium dodecyl sulfate at a flow rate of 1.0 ml/min. the drug was monitored by fluorescence detection with excitation at 347 nm and a 418 nm emission cutoff filter. Approximately 3.0 hr was required to assay an 8 point standard curve and a drug-free plasma sample. Employing 50 μl of plasma, the lowest concentration on the camptothecin lactone and total drug standard curves, 2.82 nM (0.49 ng/ml), was quantified with 3.79 and 5.58% coefficients of variation, respectively. the method has been shown to be specific and reproducible.     

High-Performance Gel-Permeation Chromatography of Bovine Skim Milk Proteins

Abstract

The separation of bovine skim milk proteins by gel-permeation high performance liquid chromatography was examined. Toya-Soda TSK-GEL (Type SW) columns were used with an eluent of .05 M phosphate buffer (pH 6.80) containing .1 M sodium sulfate at .5 ml/min. Bovine whole milk was centrifuged to remove lipids, and the resultant skim milk directly injected. A 2000SW column yielded three protein peaks: 1 = casein, IgG and BSA; 2 = 6-lactoglobulins and BSA; and 3 = α-lactalbumin and BSA. A 3000SW plus 2000SW column system with a 30 μl injection volume yielded four protein peaks: 1 = minor amounts of α - and β-casein; 2 = casein, BSA and IgG; 3 = β-lactoglobulins; and 4 = α¹-lactalbumin. A 3000SW plus 2000SW column system with a 10 μl injection volume yielded five protein peaks: 1 = casein; 2 = IgG; 3 = BSA; 4 = β-lactoglobulins; and 5 = α-lactalbumin. Both the single column and dual column applications yielded three nonprotein peaks, which were dialyzed from solution. Thus, a high speed analytical separation of milk proteins was achieved according to molecular size, but this application is highly dependent on sample size.     

High-Performance Aqueous Size-Exclusion Chromatography Using Diol-Bonded Porous Glass Packing Materials. Retention Behavior of Some Proteins

Abstract

Retention volume of proteins increased or decreased with increasing phosphate buffer or neutral electrolyte concentrations in the mobile phase. This variation suppressed or accelerated by changing pH values in the mobile phase. The behavior of proteins can be interpreted by knowing isoelectric points (pI) of proteins and pKa value of the residual silanol groups on the surface of diol-bonded porous glasses. Positively charged surface of proteins below pH 8.0 (cytochrome c, lysozyme) retarded the elution by the ion-adsorption effects and negatively charged proteins around pH 7.0 (egg albumin, bovin serum albumin) eluted earlier than expected by the ion-exclusion effects. These effects suppressed by increasing phosphate buffer and neutral electrolyte concentrations in the mobile phase. Size-exclusion separation was attained in the mobile phase over 0.1 M phosphates and 0.1 M NaCl concentrations at pH 7.0. Mcllvaine buffer and Gomori buffer showed opposite action to proteins for retention comparing with Soerensen phosphate buffer. Potassium thiocyanate showed the different action for retention of proteins comparing with other neutral electrolytes and acted like sodium dodecyl sulphonate.     

Column-switching high-performance liquid chromatographic determination of clarithromycin in human plasma with electrochemical detection

A column-switching HPLC method was described for the direct analysis of clarithromycin in human plasma using electrochemical detector without sample pre-purification step. Plasma samples were diluted with washing solvent, i.e. acetonirile-methanol-0.05 M potassium phosphate buffer (pH 7.0) (5:2:93, v/v) and then, injected to the precolumn. After plasma proteins had flowed out from the precolumn, clarithromycin and internal standard (roxithromycin) were eluted to a Luna 2 C(18) column and separated with acetonitrile-methanol-0.05 M potassium phosphate buffer (pH 7.0) (41:6:53, v/v). Electrochemical oxidation of clarithromycin occurred at 0.87 V vs. Ag/AgCl reference electrode with glassy carbon electrode. The calibration curve was linear in the concentration range 0.1-4 mug ml(-1) with correlation coefficient of 0.998. This method showed excellent precision (RSD 3.8% at 0.1 mug ml(-1)) and accuracy (+/-2%) with the total analysis time per sample of 30 min. The present method was successfully applied to the pharmacokinetic study of clarithromycin in volunteers receiving a single oral administration of clarithromycin.     

Simultaneous determination of prostaglandins E1, A1, and B1 by reversed-phase high-performance liquid chromatography for the kinetic studies of prostaglandin E1 in solution.

A method for the simultaneous determination of prostaglandins E1, A1 and B1 (PGE1, PGA1 and PGB1) in solution has been developed by reversed-phase high-performance liquid chromatography using a 3 microns C18 column. The mobile phase consisted of 35% acetonitrile in 0.002 M phosphate buffer (pH 3.5) and its flow-rate was 1.5 ml/min. Quantitative measurement was performed using a photodiode array detector system at 190, 220, and 280 nm for PGE1, PGA1 and PGB1, respectively. The method has been applied to the primary kinetic studies for reaction profile for PGE1----PGA1----PGB1 at 60 degrees C in pH 2.0, 7.2, 10.0 and 12.0 buffer solutions.     

高效液相色谱法测定染发剂中的22种染料成分

建立了染发剂中22种染料成分的高效液相色谱测定方法。使用Discovery RP-Amide C16柱(250 mm×4.6 mm,5 μm),以乙腈-0.025 mol/L磷酸盐缓冲液(pH 6.0,含0.1%的庚烷磺酸钠离子对试剂)为流动相,流速为1.0 mL/min;使用二极管阵列检测器(DAD),检测波长为260 nm和280 nm,柱温为25 ℃。该方法除了对低浓度的甲苯-2,5-二胺硫酸盐、2-甲基雷锁辛和N,N-二乙基甲苯-2,5-二胺HCl外,对其他各组分的含量测定值的相对标准偏差(RSD)均小于10%,加标回收率为77.6%～122.8%。该方法简便、准确、快速,适于氧化型染发剂中染料成分的检测。     

A simple and selective UHPLC–MS/MS method for quantification of plantagoguanidinic acid in rat plasma and its application to a pharmacokinetic study

A simple, sensitive and specific UHPLC–MS/MS method for quantification of plantagoguanidinic acid (PGA) in rat plasma was applied to investigate the pharmacokinetic behavior in vivo , using protopine as internal standard. The chromatography was separated on a Phenomenex® Luna‐C₁₈ column (2.1 × 150 mm, 3.0 μm) within 7.0 min using a mobile phase consisting of acetonitrile–0.1% formic acid solution under gradient elution at a flow rate of 0.4 mL/min. Prepared samples were monitored by multiple reaction monitoring mode, with the target fragmentions m/z 226.2 → 84.2 for PGA and m/z 354.2 → 188.9 for IS in positive electrospray ionization. The calibration curve of PGA was linear throughout the range 1–1000 ng/mL (r = 0.9962). The lower limit of quantitation in plasma for PGA was 0.1 ng/mL, and the recovery was >88.6%. Intra‐ and interday accuracy ranged from −8.6 to 4.9%. Furthermore, this validated method was successfully used for a pre‐clinical pharmacokinetic study of PGA at a single dose of 20 and 5 mg/kg in rats via oral and intravenous administration. The study showed that PGA was absorpted rapidly and eliminated gradually with a greater absolute oral bioavailability of 70.1% in rats.     

An HPLC Method for the Determination of Diltiazem and Desacetyldiltiazem in Human Plasma

Abstract

A high performance liquid chromatographic method is presented for the determination of diltiazem and its metabolite desacetyldiltiazem in human plasma. Diltiazem and desacetyldiltiazem are extracted from plasma basified with 0.5M dibasic sodium phosphate (pH 7.4) using 1% 2-propanol in n-hexane containing diazepam as an internal standard. A reversed phase cyanopropylsilane column was used with a mobile phase of 45% acetonitrile and 55% 0.05M acetate buffer (pH 4.0). The minimum detectable limit was 2ng/ml of plasma. The effect of the pH, molarity, and percent acetonitrile of the mobile phase on the capacity factor was studied. Possible interferences from other drugs administered concurrently are presented.     

Quantitative Separation of Volatile Fatty Acids by High-Pressure Liquid Chromatography

Abstract

Volatile fatty acids (acetic, propionic, butyric, isovaleric, and valeric) are separated isocratically on a reverse phase C₁₈ μBONDAPAK column in less than 20 min. The eluent was 0.01 M NaH₂PO₄ buffer, pH 3.5, containing 10% methanol. Separations were monitored by UV absorption at 210 nm. Peak height measurements gave quantitative linear responses from 0.25 μmole to 2.50 μmole of each acid.