High-pressure liquid chromatographic analysis of diazepam, oxazepam and n-desmethyldiazepam in human blood

We describe a rapid method for precisely measuring concentrations of diazepam, oxazepam and N-desmethyldiazepam in blood by high-pressure liquid chromatography. The drugs, together with an internal standard, prazepam, are extracted from 2 ml of blood and analyzed isocratically on a reversed-phase column with a mobile phase consisting of acetonitrile-0.01 M sodium acetate buffer (35:65 v/v). The eluted drugs are detected by their absorption at 240 nm. The sensitivity of this method is 30 microgram/l for oxazepam and N-desmethyldiazepam, 40 microgram/l for diazapam, for 2-ml blood samples. Relative recovery of added drugs to blood varies from 91 to 110%. The day-to-day precision (coefficient of variation) established by 10 replicate analyses was 2.8 to 9.6%.     

高效液相色谱法同时测定血清中茶碱、苯妥英钠、苯巴比妥及卡马西平的药物浓度

 报道了用高效液相色谱法（HPLC）同时测定血清中茶减、苯妥莫纳、苯巴比妥及卡马西平的药物浓度。实验条件:Nova-PahC18柱，流动相为甲醇-水（1:1，V／V），检测波长210nm，流速为1mL／min，萃取液为级访-异丙醇（95:5，V／V）。方法具有灵敏（10-9）、准确（回收率在97％～105％之间）、快速（7min）等特点，对临床血药浓度监测有实际应用价值。     

Simultaneous determination of trimethoprim,enrofloxacin, and ciprofloxacin in blood serum of poultry

A method is proposed for the simultaneous determination of trimethoprim, enrofloxacin, and ciprofloxacin in blood serum of poultry using HPLC. Samples were prepared using protein precipitation. The chromatographic separation of substances was attained on a C18 column using a mixture of a 50 mM acetate buffer solution (pH 3.0) with acetonitrile (86: 14) as a mobile phase. Detection was performed at 278 nm. Linearity was observed in the concentration range 50–5000 ng/mL for trimethoprim and 10–5000 ng/mL for ciprofloxacin and enrofloxacin. Precision was ≤2.1% for enrofloxacin, ≤1.6% for ciprofloxacin, and ≤14% for trimethoprim. Accuracy was ≤10.2% for enrofloxacin, ≤9.9% for ciprofloxacin, and ≤11.9% for trimethoprim. The method was applied to pharmacokinetic studies of complex antibacterial drugs containing trimethoprim and enrofloxacin as active substances.     

高效液相色谱法同时测定血清中茶碱、苯妥英钠、苯巴比妥及卡马西平的药物浓度

报道了用高效液相色谱法（HPLC）同时测定血清中茶减、苯妥莫纳、苯巴比妥及卡马西平的药物浓度。实验条件:Nova-PahC18柱，流动相为甲醇-水（1:1，V／V），检测波长210nm，流速为1mL／min，萃取液为级访-异丙醇（95:5，V／V）。方法具有灵敏（10-9）、准确（回收率在97％～105％之间）、快速（7min）等特点，对临床血药浓度监测有实际应用价值。     

Direct injection determination of theophylline and caffeine in blood serum by high-performance liquid chromatography using an ODS column coated with a zwitterionic bile acid derivative

An ODS column dynamically coated with zwitterionic bile acid derivative, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), was evaluated for direct injection determination of drugs in blood serum by HPLC. Polar functional groups such as sulfonate, ammonium and the three hydroxyl groups in CHAPS protruding towards an aqueous mobile phase formed a hydrophilic layer over the ODS reversed-phase surface, which resulted in high molecular mass compounds such as proteins being prevented from penetrating into the internal hydrophobic region. The bulk of the proteins were eluted as an unretained or nearly unretained band by using 0.2 mM sodium hydrogenphosphate solution (pH 7.4) as the mobile phase. In contrast, small molecules such as some inorganic anions and aromatic compounds were retained and thereby separated from one another. It was confirmed that the ODS column modified with CHAPS acts as a restricted access-type column with a hydrophobic interior and a hydrophilic exterior. Hence biological fluids could be directly injected into the CHAPS-coated ODS column. The present HPLC system using the CHAPS-coated ODS column was applied to the determination of theophylline and caffeine in human blood serum. The detection limits for the two drugs with UV absorption at 273 nm were 0.2 and 0.5 mg l-1 (injection volume 20 microliters) and the relative standard deviations of peak area measurements were < 1.4% and 2.2%, respectively, for 10 replicate measurements of serum spiked with 5 mg l-1 of each of the drugs.     

Determination of mefloquine in blood, filter paper-absorbed blood and urine by 9-fluorenylmethyl chloroformate derivatization followed by liquid chromatography with fluorescence detection

We describe a method for determination of mefloquine (MQ) in 100-microliters samples of urine, whole blood, and capillary blood collected on filter paper; quantification is by liquid chromatography with fluorescence detection at 475 nm of the 9-fluorenylmethyleneoxycarbonyl derivative. Whole blood and urine samples were prepared by extraction of MQ and internal standard from aqueous base with methyl tert.-butyl ether (MTBE), separation and evaporation of the MTBE layer, and derivatization using a solution of 9-fluorenylmethyl chloroformate in acetonitrile. Filter paper spots were immersed for 16 h in 0.1 M hydrochloric acid, followed by extraction with MTBE from aqueous sodium carbonate. The separated and evaporated organic layer was treated with the derivatizing solution. An aliquot was injected onto a high-performance liquid chromatography system using a C18 reversed-phase column and acetonitrile-water (72:28) mobile phase for filter paper spot extracts as for whole blood and urine extracts. The method has a limit of determination in blood, blood spots, and urine of 50 ng/ml with 100 microliters sample size (coefficient of variation = 16%). Linearity and precision (within-day and between-day) for the method are good. The MQ derivative was isolated and characterized spectroscopically. Values for MQ concentrations in filter paper blood spots compared favorably with values found in corresponding whole blood samples analyzed by a published method.     

Direct determination of five fluoroquinolones in chicken whole blood and in veterinary drugs by HPLC

A direct, accurate, and sensitive chromatographic analytical method for the quantitative determination of five fluoroquinolones (enoxacin, ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin) in chicken whole blood is proposed in the present study. For quantitative determination lamotrigine was used as internal standard at a concentration of 20 ng/microL. The developed method was successfully applied to the determination of enrofloxacin, as the main component of commercially available veterinary drugs. Fluoroquinolone antibiotics were separated on an Inertsil (250 x 4 mm) C8, 5 microm, analytical column, at ambient temperature. The mobile phase consisted of a mixture of citric acid (0.4 mol L(-1))-CH3OH-CH3CN (87:9:4% v/v) leading to retention times less than 14 min, at a flow rate 1.4 mL min(-1). UV detection at 275 nm provided limits of detection of 2 ng/mL per 20 microL injected volume for enoxacin, norfloxacin, and ciprofloxacin, 0.4 ng/mL for ofloxacin, and 4 ng/mL for enrofloxacin. Preparation of chicken blood samples is based on the deproteinization with acetonitrile while the pharmaceutical drug was simply diluted with water. Peaks of examined analytes in real samples were identified by means of a photodiode array detector. The method was validated in terms of within-day (n=6) precision and accuracy after chicken whole blood sample deproteinization by CH3CN. Using 50 microL of chicken blood sample, recovery rates at fortification levels of 40, 60, and 80 ng ranged from 86.7% to 103.7%. The applicability of the method was evaluated using real samples from chicken under fluoroquinolone treatment.     

超高效液相色谱-串联质谱法检验腐败血中吗啡和6-单乙酰吗啡

采用改良的QuEChERS前处理方法，建立了同时检验腐败血中吗啡和6-单乙酰吗啡的超高效液相色谱-串联质谱（UPLC-MS/MS）方法。腐败血中加入乙腈-水（4：1，v/v）混合溶液、30 mg NaCl和60 mg MgSO₄盐析促进相分离，最后经25 mg N-丙基乙二胺（PSA）和25 mg MgSO₄净化。选用ZORBAX Eclipse Plus C₁₈色谱柱分离，0.01%（v/v）氨水和乙腈作为流动相进行梯度洗脱，电喷雾电离正离子（ESI⁺）模式扫描，多反应监测（MRM）模式检测。结果表明，吗啡和6-单乙酰吗啡在5~200 μg/L范围内线性关系良好，相关系数（r²）≥ 0.9957，检出限（S/N=3）均为1 μg/L，定量限（S/N=10）均为5 μg/L。3个加标水平（5、100和200 μg/L）下，吗啡和6-单乙酰吗啡的平均加标回收率分别为81.84%~103.44%和81.03%~104.46%，日内和日间精密度（RSD）均小于12%，基质效应为83.04%~107.61%。方法简便、灵敏、可靠，可实现腐败血中吗啡和6-单乙酰吗啡的快速定性定量检验。     

Identification and quantification of 34 drugs and toxic compounds in blood,urine, and gastric content using liquid chromatography with tandem mass spectrometry

A liquid chromatography with tandem mass spectrometry method was developed for the simultaneous screening of 34 drugs and poisons in forensic cases. Blood (0.5 mL, diluted 1:1 with water) or 1.0 mL of urine was purified by solid‐phase extraction. Gastric contents (diluted 1:1 with water) were treated with acetonitrile, centrifuged, and supernatant injected. Detection was achieved using a Waters Alliance 2695/Quattro Premier XE liquid chromatography tandem mass spectrometry system equipped with electrospray ionization, operated in the multiple reaction monitoring modes. The method was validated for accuracy, precision, linearity, and recovery. The absolute recovery of drugs and toxic compounds in blood was greater than 51% with the limit of detection in the range of 0.02–20 ng/mL. The absolute recovery of drugs and toxic compounds in urine was greater than 61% with limit of detection in the range of 0.01–10 ng/mL. The matrix effect of drugs and toxic compounds in urine was 65–117% and 67–121% in blood. The limit of detection of drugs and toxic compounds in gastric content samples were in the range of 0.05–20 ng/mL. This method was applied to the routine analysis of drugs and toxic compounds in postmortem blood, urine, and gastric content samples. The method was applied to actual forensic cases with examples given.     

Automated analysis of oxolinic acid and flumequine in salmon whole blood and plasma using dialysis combined with trace enrichment as on-line sample preparation for high-performance liquid chromatography

The use of dialysis as sample clean-up for high-performance liquid chromatography makes fully automated determination of drugs in whole blood and plasma possible. High recoveries of the analytes oxolinic acid and flumequine and the internal standard nalidixic acid are obtained after a short time of dialysis (7.3 min). The dilute dialysates are enriched on a small column packed with polystyrene. When dialysis is discontinued, the analytes are eluted by mobile phase to the analytical column. With UV detection the limit of detection was 50 ng/ml for both oxolinic acid and flumequine. Validation showed good precision and accuracy and good correlation between determinations in plasma and whole blood.     

高效液相色谱法快速同时检测全血中两种免疫抑制剂

环孢素A和西罗莫司是许多器官移植手术中广泛使用的免疫抑制剂,且一起使用时会产生协同效应,但这两种免疫抑制剂的治疗窗口都非常窄,仅在特定的血药浓度范围内有预期的治疗效果。因此,快速同时检测全血中这两种免疫抑制剂的浓度,可为患者器官移植手术后的给药方案提供有价值的信息。该工作首先考察了环孢素A和西罗莫司在生物液相色谱柱和传统液相色谱柱上的色谱行为,然后基于生物液相色谱柱,建立了可快速分离和检测全血中环孢素A和西罗莫司的高效液相色谱分析方法。全血样品经样品前处理后进样分析,采用ZORBAX 300SB C8柱（250 mm×4.6 mm, 5.0 μm）进行分离,以乙腈-水（70∶30, v/v）为流动相进行等度洗脱,柱温为60 ℃,流速为1.0 mL/min,检测波长为205 nm和278 nm,进样量为20 μL。结果表明,环孢素A和西罗莫司在6 min内可实现较好的分离;环孢素A和西罗莫司在各自的浓度范围内具有良好的线性关系（r>0.997）,检出限（S/N=3）分别为10 ng/mL和1 ng/mL,定量限（S/N=10）分别为30 ng/mL和2 ng/mL, 3个水平的平均加标回收率分别为83.5%~89.7%和95.8%~97.8%,相对标准偏差（RSD）分别为3.2%~9.0%和3.4%~6.7%（n=5）。该方法操作简便,流动相简单,分析时间短,线性范围宽,灵敏度高,可用于全血中环孢素A和西罗莫司的含量检测。     

Determination of pamidronate in human whole blood and urine by reversed-phase HPLC with fluorescence detection

Pamidronate is a bisphosphonate that is effective in treating bone disease including osteopenia and osteoporosis in adults. A sensitive and reliable method for the analysis of pamidronate in whole blood and urine is key to the development of this drug for use in children. A previously described method for pamidronate analysis serum and urine did not consistently detect the drug at satisfactory levels in whole blood. The procedure involves co-precipitation of the bisphosphonates with calcium phosphate, pre-column derivitization with fluorescamine, HPLC utilizing a Nucleosil C(18) column, and fluorescence detection with excitation at 395 nm and emission at 480 nm.Changes to the original protocol included the use of a new internal standard (alendronate), the optimization of the concentration of ethylenediaminetetraacetic acid (EDTA) for dissolving the precipitate, and the elimination of the acidification step prior to deproteinization. The optimum EDTA concentration, which had a significant effect on the labeling capability of fluorescamine, was determined to be 20 mm.A good separation between pamidronate and alendronate was achieved using a heated (40 degrees C ) Nucleosil C(18), 10 micro m particle size column. The mobile phase was an aqueous solution of 1 mm Na(2)EDTA-methanol (97:3, v/v) adjusted to pH 6.5 using a fl ow-rate of 1 mL/min. Fluorescence detection was set at 395 nm for excitation and at 480 nm for emission. The limit of quantitation for pamidronate was 0.5 micro g/mL in whole blood and 0.1 micro g/mL in urine. The method was applied to both whole blood and urine samples from pediatric patients.     

Quantitative determination of porphyrins, their precursors and zinc protoporphyrin in whole blood and dried blood by high-performance liquid chromatography with fluorimetric detection

Methods for the determination of porphyrins, delta-aminolevulinic acid (ALA), porphobilinogen (PBG) and zinc protoporphyrin of heme biosynthesis in whole blood and dried blood are described. Erythrocyte porphyrins and the precursors ALA and PBG were extracted from whole blood (50 microliter) with 0.3 ml of methanol and 1.5 M hydrochloric acid (2:1, v/v). Zinc protoporphyrin was extracted with an acetone-pyridine-Sterox solution. Other major interfering metabolites were removed by centrifugation. An aliquot of the supernatant was injected onto the reversed-phase C18 column for detection of porphyrins with excitation wavelength at 405 nm and emission wavelength at 630 nm. The mobile phase was 0.1 M phosphate-methanol-tetrahydrofuran (18:30:16, v/v/v), pH 5.38. The ALA and PBG were derivatized with o-phthalaldehyde before injection. The detection excitation wavelength occurred at 330 nm and the emission wavelength at 418 nm. The mobile phase was 0.1 M phosphate-methanol (7.5:5), pH 3.38. For the dried blood specimen of filter paper, two 0.64-cm discs punched out from the blood-impregnated filter paper were placed in a test tube containing 200 microliter of 0.9% saline for 60 min or longer at room temperature and then treated as whole blood.     

On-line dialysis and weak cation-exchange enrichment of dialysate. Automated high-performance liquid chromatography of pholcodine in human plasma and whole blood.

An automated method for the determination of pholcodine in plasma and whole blood is described. The technique combines dialysis and trace enrichment prior to high-performance liquid chromatography. Dialysis, trace enrichment on a weak cation-exchange column, separation on a cyano column and fluorescence detection was shown to be an extremely selective and sensitive method. The method has been used successfully in the analysis of real samples after administration of pholcodine. The automated method can be used, after minor modification, to determine other basic drugs in whole blood and plasma.     

Bioanalytical method for the estimation of co‐administered esomeprazole,leflunomide and ibuprofen in human plasma and in pharmaceutical dosage forms using micellar liquid chromatography

The present study represents a connection between basic science and clinical applied science through providing a bioanalytical method for the analysis of certain co‐administered drugs used for the treatment of rheumatoid arthritis. The studied drugs are esomeprazole, leflunomide and ibuprofen. The proposed bioanalytical method is a simple reversed phase high performance liquid chromatographic method using micellar mobile phase. The method is conducted using a Shim‐pack VP‐ODS (150 mm × 4.6 mm ID) stainless steel column at ambient temperature with ultraviolet detection at 285 nm. The micellar mobile phase consisted of 0.1 m sodium dodecyl sulfate, 10% n‐propanol, 0.3% triethylamine in 0.02 m orthophosphoric acid (pH 3.5) and is pumped at a flow rate of 1.0 mL/min. The calibration curve was rectilinear over the concentration range of 0.1–5.0, 0.5–10.0 and 1.0–20.0 μg/mL for esomeprazole, leflunomide and ibuprofen respectively. The proposed method was successfully applied to the analysis of these drugs in dosage forms. The method is extended to the in‐vitro , in‐vivo determination of these drugs in spiked and real human plasma samples.     

Analysis of blood and urine samples from Macaca mulata for pyronaridine by high-performance liquid chromatography with electrochemical detection

We describe a high-performance liquid chromatographic method with electrochemical detection for quantifying pyronaridine in rhesus monkey (Macaca mulata) blood and urine samples. The detection limit is 20 ng/ml at a signal-to-noise ratio of 4 in 0.5-ml samples of blood or urine. Blood analysis includes a liquid-liquid extraction and a subsequent solid-phase extraction that removes an interferent present in blood. For urine, a back-extraction is substituted for the solid-phase extraction step. The method uses an analogue of amodiaquine as internal standard, a 10-microns rigid macroporous styrene-divinylbenzene copolymer column and a mobile phase of 1% (v/v) triethylamine in methanol-water (34:66, v/v). The method was applied to samples of blood and urine from a monkey after a single intramuscular dose of pyronaridine tetraphosphate (160 mg as base).     

Simultaneous determination of haloperidol and its metabolite, reduced haloperidol, in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the simultaneous determination of haloperidol and reduced haloperidol in human plasma, urine and rat tissue homogenates using bromperidol as an internal standard. The method involved extraction followed by injection of 50-80 microliters of the aqueous layer onto a C18 reversed-phase column. The mobile phase was 0.5 M phosphate buffer-acetonitrile-methanol (58:31:11, v/v/v) and the flow-rate was 0.6 ml/min. The column effluent was monitored by ultraviolet detection at 214 nm. The retention times for reduced haloperidol, haloperidol and bromperidol were 5.4, 7.2 and 8.4 min, respectively. The detection limits for haloperidol and reduced haloperidol in human plasma were both 0.5 ng/ml, and the corresponding values in human urine were both 5 ng/ml. The coefficients of variation of the assay were generally low (below 10.7%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or any drug tested were found.     

A simultaneous packed column supercritical fluid chromatographic method for ibuprofen, chlorzoxazone and acetaminophen in bulk and dosage forms

A reproducible and efficient method for the separation and estimation of ibuprofen, chlorzoxazone and acetaminophen has been developed using packed column supercritical fluid chromatography (SFC). The separations were performed on an ODS-RP JASCO column employing methanol modified supercritical fluid CO(2) as the mobile phase. The densities and polarities of the mobile phase were optimised from the effects of pressure, temperature and modifier concentration on retention times. In addition a flow programming of the mobile phase helped to obtain better resolution and a faster elution for acetaminophen. The analytes were detected using a uv detector at 254 nm. The study includes a successful attempt at quantitation of the 3 drugs. Chromatographic figures of merit, linear dynamic range, limit of quantitation (LOQ), precision and accuracy etc. were determined to assess the viability of the method. The method has been extended to commercial dosage forms containing all 3 drugs.     

Determination of bovine blood oleandrin by high-performance liquid chromatography and postcolumn derivatization

A high-performance liquid chromatographic procedure with a postcolumn fluorescence derivatization is developed for the analysis of oleandrin in bovine blood. Oleandrin is separated by an octadecylsilane-bonded column with a mobile phase containing dehydroascorbic acid. The effluent of the column is mixed with concentrated hydrochloric acid and passed through poly(tetrafluoroethylene) tubing maintained at 70 degrees C. The resultant fluorophores are detected at 465 nm with excitation at 348 nm. Simple solid-phase extraction using Sep-Pak tC2 is effective for sample purification. We found the minimal detectable quantity of oleandrin in plasma to be 1.5 ng/mL at a signal-to-noise ratio of 3:1.     

Trace analysis of benzodiazepine drugs in blood using deactivated amberlite XAD-7 porous polymer beads and silica capillary column gas chromatography with electron-capture detection

A general method for the trace analysis of benzodiazepine drugs and their major metabolites at single dose therapeutic levels in 0.2 ml blood samples is described. The method involves solvent extraction of blood with toluene, isolation of the analytes using deactivated Amberlite XAD-7 porous polymer beads, and analysis of the cleaned-up extracts by capillary column gas chromatography with electron-capture detection. The clean-up technique eliminates lipids and other interfering material, enabling routine analysis of blood extracts to be carried out with no significant deterioration in column or detector performance over a period of many months. The use of fused-silica capillary columns coated with SE-52 and the correct choice of chromatographic conditions permits underivatised benzodiazepines of widely differing volatilities and polarites to be analysed. Data for 26 benzodiazepines and metabolites are presented.