Micellar electrokinetic capillary chromatographic method for the quantitative analysis of uricosuric and antigout drugs in pharmaceutical preparations

A simple MEKC method is described for the separation and quantification of seven widely used uricosuric and antigout drugs, including allopurinol (AP), benzbromarone (BZB), colchicine (COL), orotic acid (OA), oxypurinol (OP), probenecid (PB), and sulfinpyrazone (SPZ). The drugs were separated in a BGE of borate buffer (45 mM; pH 9.00) with SDS (20 mM) as the micellar source and the separated drugs were directly monitored with a UV detector (214 nm). Several parameters affecting the separation and analysis of the drugs were studied. Based on the normalized peak-area ratios of the drugs to an internal standard versus the concentration of the drugs, the method is applicable to quantify BZB, COL, and SPZ (each 5-200 microM), AP, OA, OP, and PB (each 10-200 microM) with detection limits (S/N = 3, 0.5 psi, 5 s injection) in the range of 0.6-4.0 microM. The precision (RSD; n = 5) and accuracy (relative error; n = 5) of the method for intraday and interday analyses of the analytes at three levels (30, 120, and 180 microM) are below 4% (n = 3). The method was demonstrated to be suitable for the analysis of AP and COL in commercial tablets with speed and simplicity.     

A simple micellar electrokinetic capillary chromatographic method for the quantitative analysis of organic expectorants

A simple and rapid micellar electrokinetic capillary chromatographic method is described for the separation and quantification of five expectorant drugs, including ambroxol (AMB), bromhexine (BRM), carbocysteine, guaiacol and guaifenesin. The drugs were separated in a mixed solution of phosphate buffer (35 mM; pH 3.90) and acetonitrile (75:25, v/v) with sodium dodecylsulfate (120 mM) as the micellar source, and the separated drugs were directly monitored with UV detector (200 nm). Several key parameters affecting the separation and analysis of the drugs were studied and optimized. Based on the corrected peak-area ratios of the drugs to an internal standard (7-hydroxy-4-methyl-coumarin) versus the concentration of the drugs, the established method is applicable to quantify AMB and BRM each over 20-150 microM, carbocysteine over 100-1500 microM, guaiacol and guaifenesin each over 10-150 microM. The detection limits (S/N=3; 0.5 psi, 5 s injection) of the method for the analytes are in the range of 3.0-5.0 microM (except that of carbocysteine at 30 microM). The precision (relative standard deviation) and accuracy (relative error) of the method for the intra-day (n=3) and inter-day (n=5) analyses of the analytes at three levels are all below 4%. The method is speedy with a run time of about 6 min for the analysis of the five analytes. Application of this method to the analysis of AMB and BRM in pharmaceutical preparations or AMB in the urine of a dosed subject proved simple and effective.     

A new high-performance liquid chromatographic/electrospray ionization tandem mass spectrometric method for the simultaneous determination of cyclophosphamide, methotrexate and 5-fluorouracil as markers of surface contamination for occupational exposure monitoring

A new high-performance liquid chromatographic/electrospray ionization tandem mass spectrometric (HPLC/ESI-MS/MS) method was developed for the simultaneous quantification of 5-fluorouracil (5FU), methotrexate (MTX) and cyclophosphamide (CP) in environmental samples. These compounds, commonly used in the treatment of cancer, are recognized as genotoxic. In order to estimate the occupational exposure of hospital personnel handling these drugs, wipe samples were taken from the working surfaces and directly analyzed (with trophosphamide as internal standard) using a reversed-phase capillary column and MS/MS detection. This is the first HPLC/MS/MS method for the simultaneous determination of 5FU, MTX and CP. The present method offers high sensitivity, with detection limits of 1.1 microg l(-1) for MTX and CP and 33.3 microg l(-1) for 5FU, avoiding any sample preconcentration procedure. Rapidity, specificity, high accuracy (mean values between 92.4 and 99.9%) and precision (mean RSD values between 3.4 and 12.1%) make the method suitable for the routine determination of these three antineoplastic drugs.     

Determination of basic pharmaceuticals in human serum by hydrophilic interaction capillary electrochromatography

Hydrophilic interaction capillary electrochromatography (HI-CEC) for the determination of basic pharmaceuticals spiked in human serum is described. The organic modifier content, ionic strength, and pH value of the mobile phase as well as the applied voltage are optimized for separation and elution of these drug analytes. Excellent separation was achieved for drugs using a mobile phase composition of 80% v/v acetonitrile in 100 mM triethylamine phosphate (TEAP) buffer at pH 2.8 with column efficiencies for analytes more than 200,000 plates/m. The samples of human serum spiked with basic drugs were directly injected after a simple acetonitrile treatment. The linear range and reproducibility of these basic drugs using an external and internal standard method were compared. As a result, the reproducibility could be greatly improved by using the internal standard method. Good calibration curves with regression coefficients more than 0.998 in the range of 5-160 microg/mL were observed with the internal standard method. The limits of quantitation, based on standards with acceptable relative standard deviations (RSDs), were below 5 microg/mL. The intra- and inter-day precisions, determined as RSDs, were less than 4.57%.     

Precolumn fluorescence labeling method for simultaneous determination of hydroxyzine and cetirizine in human serum

A highly selective and sensitive method was developed for simultaneous determination of the antihistaminic drug hydroxyzine (HZ) and its pharmacologically active metabolite cetirizine (CZ) in human serum using haloperidol as internal standard. The method was based on fluorescence labeling of both drugs with a fluorescent arylboronic acid 4-(4,5-diphenyl-1H-imidazol-2-yl)phenyl boronic acid followed by separation on silica column using a mobile phase consisting of acetonitrile and water (90:10, v/v%) containing triethylamine and acetic acid. The labeling reaction conditions were optimized and the liquid-liquid extraction method was successfully applied to extract the both drugs from serum. The linearity range was 0.025-2.00 microg/mL for HZ and CZ. The limit of detection (S/N = 3) was 10 and 5 ng/mL for HZ and CZ, respectively.     

HPLC determination of diltiazem in human plasma and its application to pharmacokinetics in humans

A simple and sensitive reversed-phase high performance liquid chromatographic method (HPLC) has been developed and validated for the routine analysis of diltiazem in human plasma and the study of the pharmacokinetics of the drug in the human body. Diltiazem and diazepa (internal standard) were extracted with a mixed organic solution of hexane, chloroform and isopropanol (60:40:5, v/v/v), and then HPLC separation of the drugs was performed on an Spherisorb C(18) column and detected by ultraviolet absorbance at 239 nm. The use of methanol-water solution (containing 2.8 mm triethylamine, 80:20, v/v) as the mobile phase at a fl ow-rate of 1.2 mL/min enables the baseline separation of the drugs free from interferences with isocratic elution. The method was linear in the clinical range 0-300 ng/mL and the lower limit of detection of diltiazem in plasma was 3 ng/mL. The range of percentage of relative standard deviation (%RSD) was from 3.5 to 6.8% for within-day analyses and from 6.2 to 8.4% for between-day analyses, respectively. The extraction recoveries of diltiazem from spiked human plasma (n = 5) at three concentrations were 91.4-104.0%. The method has been used to determine diltiazem in human plasma samples from eight volunteers who had taken diltiazem hydrochloride slow release tables and the data obtained was fitted with a program on computer to study the pharmacokinetics. The results showed that the peak level in plasma approximately averaged 118.5 +/- 14.3 ng/mL at 3.1 +/- 0.4 h, and the areas under the drug concentration curves (AUC) was 793.1 +/- 83.1 ng.h/mL.     

同位素稀释超高效液相色谱串联质谱法同时测定牛奶中的克伦特罗、氯霉素与己烯雌酚

建立了同时测定牛奶中克伦特罗、氯霉素和己烯雌酚残留量的同位素稀释超高效液相色谱-串联质谱(UPLC-MS/MS)分析方法.牛奶样品无需蛋白沉淀,直接经HLB小柱净化及水和正己烷淋洗,由乙酸乙酯洗脱后进行分析.采用Acquity UPLC(○R)BEH C18色谱柱进行分离,以乙酸铵溶液-乙腈作为流动相进行梯度洗脱,MR...     

超高效液相色谱-串联质谱法快速测定鱼和虾中多类禁、限用兽药残留

建立了鱼和虾中5类（磺胺类、喹诺酮类、氯霉素类、硝基呋喃类代谢物、三苯甲烷类）28种禁、限用兽药的同时提取净化-超高效液相色谱-串联质谱（UHPLC-MS/MS）快速检测的方法。对样品前处理及色谱条件进行优化,以盐酸水解样品,待硝基呋喃类代谢物与2-硝基苯甲醛衍生化后,5类残留药物经乙腈提取,提取液经氯化钠脱水、盐析,乙腈层经正己烷净化后浓缩至近干,残留物用甲醇定容至1.0 mL,加50 mg C18、30 mg N-丙基乙二胺（PSA）、60 mg氨基键合硅胶填料（NH2）吸附剂分散固相萃取净化,最后经ZORBAX C18色谱柱（50 mm×2.1 mm,1.8 μm）分离,在正、负离子多反应监测模式下采集,同位素内标法定量。所建立的方法确保28种兽药在各自范围内线性关系良好,相关系数（r）>0.99,方法的检出限和定量限分别为0.1~0.8 μg/kg和0.3~2.7 μg/kg;在1、5、20倍定量限添加水平下的平均回收率为70.0%~120%,相对标准偏差为0.9%~10.0%。方法简便、快速、准确、灵敏,适用于鱼和虾中多类禁、限用药物的快速测定。     

High performance liquid chromatography-high resolution mass spectrometry and micropulverized extraction for the quantification of amphetamines, cocaine, opioids, benzodiazepines, antidepressants and hallucinogens in 2.5 mg hair samples

A high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) method for simultaneous screening and quantification of 28 drugs was developed and validated for 2.5 mg hair samples. Target drugs and their metabolites included amphetamines, cocaine, opioids, benzodiazepines, antidepressants, and hallucinogens. After decontamination, hair samples were extracted with 200 μL of a mixture of water: acetonitrile:1 M trifluoroacetic acid (80:10:10, v/v) using a 5 min simultaneous pulverization/extraction step. The extracts were analysed by HPLC-HRMS in an Orbitrap at a nominal resolution of 60,000, with concomitant in source collisional experiments (in source CID). Gradient elution on an Atlantis T3 column resolved 28 target compounds and 5 internal standards. Total chromatographic run time was 26 min. Calibration was achieved by linear regression analysis utilizing six calibration points; R2 ranged from 0.9964 to 0.9999, the limits of quantification were 0.1 ng/mg for 8 compounds, 0.2 ng/mg for 16 compounds and 0.5 ng/mg for 4 compounds; mean relative errors from -21% to +23% were obtained; relative standard deviation, used to estimate repeatability and intermediate reproducibility at three concentrations, was always less than 20%. Process efficiency and recoveries for all analytes were better than 65 and 73%, respectively, at any concentration. The method was applied to hair samples from forensic investigations that contained a broad assortment of drugs of abuse and pharmaceuticals. The use of concomitant HRMS full scan and CID afforded the possibility of retrospective analysis for discovering untargeted drugs.     

高效液相色谱-质谱联用法检测免疫抑制剂及其合并用药

建立高效液相色谱-质谱联用法同时测定人血浆中免疫抑制剂及合并用药12种药物浓度的方法.选用Kromasil-C18色谱柱(50 mm× 4.6 mm×5 μm),以甲醇-乙腈-1mmol/L乙酸铵溶液为流动相,采用梯度洗脱进行分离,样本用甲醇沉淀蛋白后进样,流速:1.1 mL/min;柱温:35℃;进样量:20μL.选用3200QTrap型液相色谱-串联质谱仪的多反应监测(MRM)扫描方式进行检测.12种药物的线性范围为0.2～1000μg/L;定量下限为0.2 μg/L.准确度与精密度结果显示方法日间、日内RSD均小于15％;相对偏差-13％～9.33％,稳定性较好.本方法快速、灵敏,专属性强、重现性好,可用于人体血浆中免疫抑制剂及其常用合并用药共12种药物浓度的测定.     

QuEChERS-超高效液相色谱-串联质谱法同时检测猪肉中121种兽药

建立了QuEChERS结合超高效液相色谱-串联质谱(UPLC-MS/MS)同时检测猪肉中 β -激动剂类、氯霉素类、阿维菌素类、磺胺类、氟喹诺酮类、硝基咪唑类、头孢类、四环素类、大环内酯类和聚醚类等10余类121种兽药的分析方法。样品经Na₂EDTA-McIlvaine缓冲溶液(pH=4)和乙腈提取,无水硫酸钠盐析,上清液分为两组,分别用不同的QuEChERS净化剂净化。使用电喷雾离子(ESI)源,在多反应监测(MRM)扫描模式下检测。5种兽药采用内标法定量,其余116种兽药采用外标法定量。121种兽药在0.02~40 μg/L范围内线性关系良好(r>0.99),定量限(S/N=10)为0.05~10 μg/kg。LOQ加标水平下,121种兽药中8种兽药的回收率为41.7%~59.6%,相对标准偏差为1.7%~13.5%; 10种兽药的回收率为122.6%~163.2%,相对标准偏差为0.6%~14.8%;其余103种兽药的回收率为60.3%~118.3%,相对标准偏差为0.4%~16.7%。该方法可对性质差别大的多类别兽药同时进行分析,在节约成本和保证检测周期方面具有突出的优势。     

分散固相萃取净化-超高效液相色谱-串联质谱法同时测定鱼和虾中抗生素及三苯甲烷类兽药残留

建立了分散固相萃取净化-超高效液相色谱-串联质谱法快速测定鱼和虾中3类（磺胺类、喹诺酮类、氯霉素类）18种抗生素及2种三苯甲烷类（孔雀石绿、隐色孔雀石绿）兽药残留。样品经磷酸氢二钾溶液水解分散，乙腈提取，提取液经氯化钠脱水、盐析后取乙腈层，经旋转蒸发仪浓缩至近干，残留物用甲醇定容至1.0 mL，C18和PSA（N-丙基乙二胺）填料分散固相萃取净化，过滤膜后经ZORBAX C18色谱柱（50 mm×2.1 mm，1.8 μm）分离，正、负离子多反应监测模式采集数据，同位素内标法定量。实验结果表明，20种兽药在0.2~300 μg/L范围内具有良好的线性关系，检出限为0.1~0.6 μg/kg，定量限为0.3~1.8 μg/kg，相关系数均大于0.99。在1、5和20倍定量限加标浓度水平下，平均回收率为72.5%~118%，相对标准偏差（RSD）为1.9%~9.8%。该方法具有前处理简单、检测效率高、成本低等优点，适用于鱼和虾中多类兽药残留的同时测定。     

Spectrophotometric and spectrofluorimetric methods for analysis of tramadol, acebutolol and dothiepin in pharmaceutical preparations

Sensitive spectrophotometric and spectrofluorimetric methods are described for the determination of tramadol, acebutolol and dothiepin (dosulepin) hydrochlorides. The two methods are based on the condensation of the cited drugs with the mixed anhydrides of malonic and acetic acids at 60 degrees C for 25-40 min. The coloured condensation products are suitable for the spectrophotometric and spectrofluorimetric determination at 329-333 and 431-434 nm (excitation at 389 nm), respectively. For the spectrophotometric method, Beer's law was obeyed from 0.5 to 2.5 microg ml(-1) for tramadol, dothiepin and 5-25 microg ml(-1) for acebutolol. Using the spectrofluorimetric method linearity ranged from 0.25 to 1.25 microg ml(-1) for tramadol, dothiepin and 1-5 microg ml(-1) for acebutolol. Mean percentage recoveries for the spectrophotometric method were 99.68+/-1.00, 99.95+/-1.11 and 99.72+/-1.01 for tramadol, acebutolol and dothiepin, respectively and for the spectrofluorimetric method, recoveries were 99.5+/-0.844, 100.32+/-0.969 and 99.82+/-1.15 for the three drugs, respectively. The optimum experimental parameters for the reaction has been studied. The validity of the described procedures was assessed. Statistical analysis of the results has been carried out revealing high accuracy and good precision. The proposed methods were successfully applied for the determination of the selected drugs in their pharmaceutical preparations with good recoveries. The procedures were accurate, simple and suitable for quality control application.     

Determination of chlorpheniramine maleate and tincture ipecac in dosage form by liquid chromatography with ultraviolet detection

A procedure was developed and validated for measuring chlorpheniramine maleate and tincture ipecac (as emetine hydrochloride) by reversed-phase liquid chromatography with methanol-10 mM sodium heptanesulfonate (20 + 30) as the mobile phase; the pH was adjusted to 4 with acetic acid, and the flow rate was at 1.5 mL/min, with ultraviolet detection at 254 nm. Propyl paraben was used as the internal standard. The standard curves were linear (r = 0.998 and 0.9998) for both chlorpheniramine maleate and emetine hydrochloride over the ranges of 5-100 and 0.1-40 microg/mL, respectively. The mean recoveries +/- standard deviation were 101.37 +/- 2.77% for chlorpheniramine maleate and 98.8 +/- 1.47% for emetine hydrochloride. The proposed method was applied to the determination of chlorpheniramine maleate alone in tablet and syrup dosage forms. The method also was applied to the determination of the emetine content of ipecac liquid extract and tincture ipecac; the results were compared with those of the method of the British Pharmacopoeia. The proposed method was applied successfully to the simultaneous determination of chlorpheniramine maleate and tincture ipecac, as emetine hydrochloride, in syrup dosage form. Both drugs and the internal standard were separated from all interfering components in < 5 min. The proposed method is simple, specific, and economical, when compared with other published methods that determine each component alone.     

Enantioselective drug monitoring of (R)- and (S)- propranolol in human plasma via derivatization with optically active (R,R)-O,O-diacetyl tartaric acid anhydride

A sensitive high-performance liquid chromatographic method was developed for the stereoselective assay of (R)- and (S)-propranolol in human plasma. The method involves diethyl ether extraction of the drugs and a racemic internal standard, N-tert.-butylpropranolol, followed by derivatization of the compounds with the chiral reagent (R,R)-O,O-diacetyl tartaric acid anhydride. The resulting diastereomeric derivatives were separated isocratically on a reversed-phase column. Quantitation was achieved by the peak-height ratio method with reference to the internal standard. The assay was accurate and reproducible in the concentration range 1-100 ng of (R)- and (S)-propranolol per ml plasma, using fluorescence detection at lambda ex 290 nm and lambda em 335 nm. The applicability of this method was demonstrated for the determination of concentration-time profiles of propranolol enantiomers in the course of comparative pharmacokinetic studies.     

Rapid ultraviolet monitoring of multiple psychotropic drugs with a renewable microfluidic device

A rapid method for sensitive ultraviolet detection of multiple psychotropic drugs in human plasma was developed on a low-cost and expediently fabricated hybrid microfluidic device. The device was composed of one fused-silica capillary with a sampling fracture, a poly(methyl methacrylate) board with four reservoirs, and a printed circuit board. At the optimal separation and detection conditions, the baseline separation of three kinds of psychotropic drugs including barbiturates (phenobarbital and barbital), benzodiazepines (nitrazepam, clonazepam, chlordiazepoxide, alprazolam and diazepam) and tricyclic antidepressant drugs (amitriptyline) was achieved within 200 s with separation efficiency up to 3.80 × 10(5) plates m(-1). The linear ranges for ultraviolet detection were from 2.0 to 1000.0 μg mL(-1) for chlordiazepoxide and 1.0 to 1000.0 μg mL(-1) for other seven drugs. Combining with solid-phase extraction, this novel protocol could successfully be used to screen naturally existing psychotropic drugs in a known human plasma sample. The minimum detectable concentration was down to 27 ng mL(-1) for phenobarbital spiked in plasma. This work provided a promising way to initially screen different psychotropic drugs with high resolution, rapid separation and low-cost.     

Simultaneous determination of cefuroxime axetil and ornidazole in tablet dosage form using reversed-phase high performance liquid chromatography

 A simple, accurate and sensitive reversed-phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous determination of cefuroxime axetil and ornidazole in combined tablet dosage form has been developed. The method was performed with a HiQ-SiL C18 column (250 mm×4.6 mm) and photodiode array (PDA) detector, using 0.01 mol/L potassium dihydrogen orthophosphate-methanol (56∶44, v/v) as the mobile phase and tinidazole as the internal standard. Beer’s law obeys in the concentration ranges of 5-25 μg/mL and 10-50 μg/mL for cefuroxime axetil and ornidazole, respectively. The method has been successfully validated statistically and applied for the analysis of the drugs in pharmaceutical formulation.     

Sol-gel titania-coated needles for solid phase dynamic extraction-GC/MS analysis of desomorphine and desocodeine

Novel sol-gel titania film coated needles for solid-phase dynamic extraction (SPDE)-GC/MS analysis of desomorphine and desocodeine are described. The high thermal stability of titania film permits efficient extraction and analysis of poorly volatile opiate drugs. The influences of sol-gel reaction time, coating layer, extraction and desorption time and temperature on the SPDE needle performance were investigated. The deuterium labeled internal standard was introduced either during the extraction of analyte or directly injected to GC after the extraction process. The latter method was shown to be more sensitive for the analysis of water and urine samples containing opiate drugs. The proposed conditions provided a wide linear range (from 5-5000 ppb), and satisfactory linearity, with R(2) values from 0.9958 to 0.9999, and prominent sensitivity, LOQs (1.0-5.0 ng/g). The sol-gel titania film coated needle with SPDE-GC/MS will be a promising technique for desomorphine and desocodeine analysis in urine.     

Liquid chromatography-tandem mass spectrometry for the determination of a new oxazolidinone antibiotic DA-7867 in human plasma

A liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the determination of a new ox-azolidinone antibiotic DA-7867, (S)-[N-3-(4-(2-(1-methyl-5-tetrazolyl)-pyridine-5-yl)-3- fl uorophenyl)-2-oxo-5-oxazolidinyl]methyl acetamide, in human plasma was developed. DA-7867 and internal standard, linezolid, were extracted from human plasma with ethyl acetate at acidic pH. A reverse-phase LC separation was performed on Luna C(8) column with the mixture of acetonitrile-ammonium formate (10 mm, pH 4.5; 35:65, v/v) as mobile phase. The analytes were determined using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The lower limits of quanti fi cation for DA-7867 was 2.5 ng/mL. The single liquid-liquid extraction quantitatively recovered DA-7867 and internal standard from plasma samples at the ranges of 82.2-86.7%. DA-7867 was stable in blank human plasma at room temperature for 24 h and following three freeze-thaw cycles.     

Chemometric determination of naproxen sodium and pseudoephedrine hydrochloride in tablets by HPLC

A new chemometric determination by high-performance liquid chromatography (HPLC) with photodiode array (PDA) detection was implemented for the simultaneous determination of naproxen sodium and pseudoephedrine hydrochloride in tablets. Three chemometric calibration techniques, classical least squares (CLS), principle component regression (PCR) and partial least squares (PLS) were applied to the peak area at multiwavelength PDA detector responses. The combinations of HPLC with chemometric calibration techniques were called HPLC-CLS, HPLC-PCR and HPLC-PLS. For comparison purposes the HPLC method called the classic HPLC method was used to confirm the results obtained from combined HPLC-chemometric calibration techniques. A good chromatographic separation between two drugs with losartan potassium as an internal standard was achieved using a Waters Symmetry C18 Column 5 microm 4.6+/-250 mm and a mobile phase containing 0.2 M acetate buffer and acetonitrile (v/v, 40:60). The multiwavelength PDA detection was measured at five different wavelengths. The chromatograms were recorded as a training set in the mobile phase. Three HPLC-chemometric calibrations and the classic-HPLC method were used to test the synthetic mixtures of naproxen sodium and pseudoephedrine hydrochloride in the presence of the internal standard. The HPLC-chemometric approaches were applied to real samples containing drugs of interest. The experimental results obtained from HPLC-chemometric calibrations were compared with those obtained by a classic HPLC method.