液相色谱-串联质谱法同时检测饲料中7种精神类药物

建立了液相色谱-串联质谱同时检测饲料样品中7种精神类药物(硝西泮、奥沙西泮、氯丙嗪、异丙嗪、地西泮、奋乃静、硫利达嗪)的方法.通过对提取溶剂、净化等预处理条件及LC-MS/MS 分析条件的优化,可以同时检测饲料中7种违禁精神类药物.饲料样品经乙腈/水(9:1, V/V)提取后,过MCX固相萃取柱净化,氮吹至干,用1 mL乙腈/水(2:8, V/V)溶解后测定,采用SRM模式进行定性与定量分析.7种精神类药物在饲料中的回收率为53.9%～110.2%; 相对标准偏差为3.4%～18.4%;硝西泮、奥沙西泮、氯丙嗪、异丙嗪的检出限为1.0 ng/g;对地西泮、奋乃静、硫利达嗪的检出限为5.0 ng/g.结果表明,本方法可用于饲料中7种精神类药物的测定.     

Development of a simple liquid chromatography-tandem mass spectrometry method for multiresidue determination of antifungal drugs in chicken tissues

A method involving LC coupled with MS/MS (LC/MS/MS) was designed for simultaneous quantification of 10 antifungal drugs (voriconazole, griseofulvin, clotrimazole, bifonazole, econazole, ketoconazole, itraconazole, miconazole, terconazole, and fluconazole) in the liver and muscles of chickens. Homogenized tissue samples were extracted with acetonitrile and subsequently underwent freezing-delipidation. A Waters Acquity Ultra Performance LC BEH C18 column was used to separate the analytes, coupled with MS/MS using an electrospray ionization source. The accuracy of the method was confirmed with a mean recovery of 71-121%, and acceptable coefficients of variation (4-23%, n = 6). The detection capability of these compounds in two different matrixes was 0.50-2.82 microg/kg. This method can be applied for the screening and confirmation of target antifungal drugs in chicken tissues.     

A high performance liquid chromatographic method for the screening of 17 diuretics in human urine

A simple and adequate HPLC method was developed for screening of human urine for the following 17 diuretic drugs: acetazolamide, bendrofluazide, bumetanide, canrenoic acid, chlorothiazide, chlorthalidone, clopamide, epitizide, etacrynic acid, furosemide, hydrochlorothiazide, indapamide, mefruside, piretanide, spironolactone, torasemide, and triamterene. The assay involves extraction from two 2 mL urine samples with ethyl acetate at pH = 5, washing with a phosphate buffer at pH = 6 and analysis by HPLC using a reversed phase C18 column and ultraviolet detection with a diode array detector for all drugs (except triamterene) using two eluents consisting of water, triethylamine, phosphoric acid and acetonitrile at different ratios and different pH values. Triamterene is determined by direct injection of diluted urine onto the column and is measured by fluorescence detection. The recoveries of the diuretic drugs were determined at two different concentrations and ranged from 43–110% (median: 87%) which is sufficient to detect abuse of these drugs. The repeatability of the assay ranged from 1–12% (median: 5.5%). Received: 13 July 1998 / Revised: 23 October 1998 / Accepted: 29 October 1998     

超高效液相色谱-四极杆/静电场轨道阱高分辨质谱快速筛查水产及水产加工品中24种镇静剂类药物

建立了超高效液相色谱-四极杆/静电场轨道阱高分辨质谱（UPLC-Q-Orbitrap HRMS）同时检测水产及水产加工品中24种镇静剂类药物的分析方法。样品经乙腈提取,浓缩近干后,用50%（v/v）甲醇水溶液定容,再用乙腈饱和的正己烷溶液净化。采用ACQUITY UPLC^® BEH C₁₈色谱柱（100 mm×2.1 mm,1.7 μm）分离,以0.1%（v/v）甲酸水溶液和含0.1%（v/v）甲酸的乙腈溶液为流动相进行梯度洗脱,在可加热电喷雾离子（HESI）源正离子模式和全扫描/数据依赖二级扫描（Full MS/dd-MS²,Top1）模式下检测,外标法定量。结果表明,24种镇静剂在各自的范围内线性关系良好,决定系数（r²）≥0.9968;在6种水产及水产加工品基质中进行3个不同添加水平的加标回收试验,24种镇静剂类药物的平均回收率为58.9%~122.9%,相对标准偏差为0.1%~16.4%（n=6）;24种镇静剂类药物的定量限为0.1~5.0 μg/kg。该法操作简便,精密度高,准确性好,适用于批量水产及水产加工品中24种镇静剂类药物的快速筛查。     

Ion-Paired Liquid Chromatographic Determination of Cefazolin in Canine Serum and Tissues

Abstract

Cefazolin is commonly used as a prophylactic antibiotic in dogs undergoing total hip arthroplasty.

A sensitive high-performance liquid chromatographic method was developed for the determination of cefazolin in canine serum and tissues. The tissues were those in contact with the hip prothesis; namely, the coxofemoral joint capsule, cancellous bone from the acetabulum and bone marrow from the femoral canal.

The method involved filtration of diluted serum or tissue extracted with ethanol:acetonitrile:water (40:10:50) through a 30,000 molecular weight cut-off filter. Separation of cefazolin from other components was by ion-paired (dodecanosulfonate) high performance liquid chromatography using a reversed-phase column eluted with acetonitrile/water solution. The ultraviolet absorbance of the column effluent was monitored at 230 nm. Recovery of cefazolin spiked at 10 μg/ml from serum was 89.8% with a coefficient of variation (CV) of 5.3% (n=10). Recovery of cefazolin spiked at 5 μg/ml from tissue extracts of joint capsule, cancellous bone and bone marrow was: 93.9%, 98.2%, and 104.2% respectively, with a CV of 6.7%, 10.2% and 10.6% respectively (n=10). A correlation coefficient of 0.9999 occurred with cefazolin in aqueous solution (n=5). The limit of detection for cefazolin was approximately 10 ng/ml of serum or tissue extract.     

A high performance liquid chromatographic method for the screening of 17 diuretics in human urine

A simple and adequate HPLC method was developed for screening of human urine for the following 17 diuretic drugs: acetazolamide, bendrofluazide, bumetanide, canrenoic acid, chlorothiazide, chlorthalidone, clopamide, epitizide, etacrynic acid, furosemide, hydrochlorothiazide, indapamide, mefruside, piretanide, spironolactone, torasemide, and triamterene. The assay involves extraction from two 2 mL urine samples with ethyl acetate at pH = 5, washing with a phosphate buffer at pH = 6 and analysis by HPLC using a reversed phase C18 column and ultraviolet detection with a diode array detector for all drugs (except triamterene) using two eluents consisting of water, triethylamine, phosphoric acid and acetonitrile at different ratios and different pH values. Triamterene is determined by direct injection of diluted urine onto the column and is measured by fluorescence detection. The recoveries of the diuretic drugs were determined at two different concentrations and ranged from 43–110% (median: 87%) which is sufficient to detect abuse of these drugs. The repeatability of the assay ranged from 1–12% (median: 5.5%).     

C4馏分中微量乙腈的大口径毛细管色谱测定

采用水相萃取富集技术，利用大口径厚液膜HLZ－60石英毛细管色变柱对C4烃抽余液中的微量乙腈进行分析；结果表明LZP－60石英毛细管色谱柱对乙腈化合物具有良好的惰性和色谱选择性；残余烃杂质峰和大量水分不干扰乙腈峰的流出；定量分析结果表明，样品测量的相对标准偏差为15％，其相对误差为7％，色谱分离检测法完全满足实际工作中微量乙腈分析的需要。     

Hydrophilic interaction liquid chromatography/electrospray mass spectrometry determination of acyclovir in pregnant rat plasma and tissues

Reversed-phase chromatography is the most common means of separation for small drug molecules. However, polar drugs may suffer from poor retention and peak shape in reversed-phase high-performance liquid chromatography (RP-HPLC). Hydrophilic interaction liquid chromatography (HILIC) provides a viable alternative to RP-HPLC and is an excellent way to separate polar compounds. This paper describes a HILIC/ESI-MS/MS assay for the determination of acyclovir from rat plasma, amniotic fluid, placental tissue, and fetal tissue. The isocratic separation utilizes an underivatized silica column with an acetonitrile/formate buffer mobile phase (80:20). The method is validated over a range of 50 ng/mL to 50 micro g/mL with % error and % relative standard deviation of <15% over 3 days. All samples are prepared by acetonitrile protein precipitation, which yields high recovery (>84% for acyclovir). This assay can be applied to the pharmacokinetic study of the placental transfer of acyclovir.     

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.     

High-performance liquid chromatographic assay for the determination of novel triazole antifungal agents in tissue. Application to tissue distribution studies

A simple and rugged reversed-phase high-performance liquid chromatographic method with ultraviolet absorbance detection at 263 nm was developed and validated for the analysis of novel triazole antifungal agents SYN-2869 and its derivatives in tissues. The method involved homogenization with 0.01 M phosphate buffer (pH 7.8) for lung, brain and spleen tissues. The liver and kidneys were homogenized with acetonitrile:acetone (1:1). The plasma proteins were precipitated with ice-cold acetonitrile and supernatent was evaporated to dryness. The reconstituted samples were injected onto an HPLC system. SYN-2869 was separated from the matrix components on a symmetry C(18) column using a aqueous mobile phase of acetonitrile and water with a flow rate of 1 mL/min. A step gradient of 40-80% acetonitrile eluted SYN-2869 and the internal standard (SYN-2506). The linear range was 0.5-10 microgram/g (r(2) > 0.99). The limit of quantitation was 0.5 microgram/g. The inter-day precision and accuracy for SYN 2869 standard concentration were from 2.6 to 7.4% and from -1.56 to +3.29%, respectively. The method was applied to tissue samples collected from single intravenous administration to mice to evaluate the distribution of these novel antifungal agents to different tissues.     

Determination of chlorpyrifos and its metabolites in rat brain tissue using coupled-column liquid chromatography/electrospray ionization tandem mass spectrometry

A method has been developed to quantify chlorpyrifos (O,O-diethyl-O-[3,5,6,-trichloro-2-pyridyl] phosphorothionate) and its metabolites chlorpyrifos-oxon (O,O-diethyl-O-[3,5,6,trichloro-2-pyridinyl] phosphate) and TCP (3,5,6,-trichloro-2-pyridinol) in rat brain tissue by coupled-column liquid chromatography/electrospray ionization tandem mass spectrometry (LC/LC/ESI-MS/MS). Rat brains were homogenized and treated by protein precipitation using ice-cold acetonitrile. The supernatant was directly injected onto the coupled-column system. Sample clean-up was achieved on a Zorbax Extend-C(18) column (2.1 x 50 mm, 5 microm) using a mobile phase of acetonitrile/water with 0.0025% formic acid (40:60, v/v). The compounds were separated isocratically on a Zorbax Eclipse XDB C(8) column (2.0 x 150 mm, 5 microm) using a mobile phase of acetonitrile/water with 0.0025% formic acid (75:25, v/v). Chlorpyrifos and chlorpyrifos-oxon were detected in positive ion mode using multiple reaction monitoring (MRM). TCP was detected in negative ion mode using precursor-to-precursor transition monitoring. The method was validated and the specificity, linearity, limit of quantitation (LOQ), precision, accuracy, stability, and recoveries were determined. Calibration curves for all three analytes yielded correlation coefficients of 0.993 or greater. The LOQs were 25.3 ng/g for chlorpyrifos and 6.3 ng/g for chlorpyrifos-oxon and TCP. All precision relative standard deviations (RSDs) were less than 16% for the LOQ and less than 11% for the other QC samples. This method was successfully applied to six rats that were injected subcutaneously with chlorpyrifos.     

Establishment of pressurized liquid extraction followed by HPLC–MS/MS method for the screening of adrenergic drugs,steroids, sedatives,colorants and antioxidants in swine feed

A facile and sensitive multi‐residue detection approach of pressurized liquid extraction following high‐performance liquid chromatography tandem mass spectrometry was established to detect the residues of adrenergic drugs, steroids, sedative, colorant and antioxidant in feed. The conditions employed for pressurized liquid extraction involved acetonitrile/ethyl acetate (1:1, v/v) as the extracting solvent, the temperature 80°C, two cycles and a static time of 10 min. The extraction was followed by a solid‐phase extraction clean‐up step. The separation of samples was done by C₁₈ column with the mobile phase of 5 mM ammonium acetate solution and acetonitrile with 0.1% formic acid. The limits of quantification ranged from 0.03 to 1 μg/kg, limits of detection were in a range of 0.01–0.5 μg/kg, and average recoveries were 70.4–98.6%. The pressurized liquid extraction procedure was optimized and overall method was validated in terms of sensitivity, linearity, selectivity, matrix effect, accuracy, recovery and stability of the target drugs in the pressurized liquid extraction extracts solution. The screening method was proved to be fast, selective, accurate and sensitive for screening drugs.     

液相色谱-串联质谱法快速筛查减肥类中药及保健品中非法添加30种化学药

建立了中药及保健品中30种减肥类化学药的高效液相色谱-串联质谱(LC-MS/MS)快速筛查方法。实验优化了前处理方法、色谱分离条件和质谱参数。样品采用甲醇超声萃取,Agilent poroshell 120 ECC18(4.6 mm×100 mm,2.7μm)色谱柱分离,流动相为乙腈-水溶液(含10 mmol/L甲酸铵,0.1%甲酸,10%甲醇),梯度洗脱,流速0.25 m L/min,采用正负离子切换模式的电喷雾质谱检测,多反应选择离子监测(MRM)。该方法能同时筛查30种化学药,覆盖面广,简便、快速、准确可靠,已用于减肥类中药及保健品中非法添加化学药的筛查及检测。     

A liquid chromatography method using a monolithic column for the determination of corticoids in animal feed and animal feeding water

An HPLC-DAD method for determining corticoids in calf feed and in animal feeding water samples using a monolithic column has been developed and validated. The method optimization included the study of binary mobile phases of water and acetonitrile. The optimum separation was achieved at 40 °C, with acetonitrile:H₂O 29:71 v/v used as mobile phase and a 3 ml/min flow-rate, which resulted in their separation in about 5 min. Two reported sample procedures were applied to feed and for animal feeding water samples prior to HPLC. Method validation was carried out according to the EU criteria established for quantitative screening methods. The results indicate that this method is highly specific, reproducible and accurate. The proposed method was found to be robust and unaffected by small variations in the extraction procedure and in HPLC conditions. The developed method for the determination of corticoids in feed and water samples was also found to be suitable for different kinds of feeds and waters.     

Multiclass method for the quantification of 92 veterinary antimicrobial drugs in livestock excreta,wastewater, and surface water by liquid chromatography with tandem mass spectrometry

A simple multiresidue method was developed for detecting and quantifying 92 veterinary antimicrobial drugs from eight classes (β‐lactams, quinolones, sulfonamides, tetracyclines, lincomycins, macrolides, chloramphenicols, and pleuromutilin) in livestock excreta and water by liquid chromatography with tandem mass spectrometry. The feces samples were extracted by ultrasound‐assisted extraction with a mixture of acetonitrile/water (80:20, v/v) and edetate disodium, followed by a cleanup using solid‐phase extraction with an amino cartridge. Water samples were purified with hydrophilic–lipophilic balance solid‐phase extraction column. Urine samples were extracted with acetonitrile and edetate disodium. Detection of veterinary antimicrobial drugs was achieved by liquid chromatography with tandem mass spectrometry using both positive and negative electrospray ionization mode. The recovery values of veterinary antimicrobial drugs in feces, urine, and water samples were 75–99, 85–110, and 85–101% and associated relative standard deviations were less than 15, 10, and 8%, respectively. The limits of quantification in feces, urine, and water samples were 0.5–1, 0.5–1, and 0.01–0.05 μg/L, respectively. This method was applied to determine real samples obtained from local farms and provides reliable quantification and identification results of 92 veterinary antimicrobial drugs in livestock excreta and water.     

Liquid chromatographic method for enantiopurity control of alaptide using polysaccharide stationary phases

Separation of veterinary drug alaptide ((S)-8-methyl-6,9-diazaspiro(4,5)decane-7,10-dione) from a chiral impurity (R-enantiomer) was developed. Five chiral columns (three amylose and two cellulose type) were evaluated in a reversed-phase system. Three of them offered satisfactory enantiomeric resolution. Finally, three methods were validated and proved to be applicable for the determination of a chiral impurity content below 0.1% (method A: 3-AmyCoat column, tris-[3,5-dimethylphenyl]carbamoyl amylose; mobile phase: water/methanol/propan-2-ol/butan-2-ol=75:20:3.5:1.5 v/v, flow rate: 0.40 mL/min; column temperature: 30 °C; method B: Chiralpak AS-3R, tris-[1-phenylethyl]carbamoyl amylose; water/acetonitrile=80:20 v/v, 0.40 mL/min; 40 °C; method C: Chiralcel OZ-3R, tris-[3-chloro-4-methylphenyl] carbamoyl cellulose; water/acetonitrile=80:20 v/v, 0.40 mL/min; 40 °C). Some decrease in efficiency with repeated sample injections was observed for the 3-AmyCoat column. The resistance to mass transfer in the stationary phase increased probably due to the change in chiral selector conformation. This effect was considerably suppressed by propan-2-ol or to a greater extent by butan-2-ol added to a mobile phase. Simple regeneration was also suggested to recover efficiency of the column.     

Validated HPLC and HPTLC Method for Simultaneous Quantitation of Amlodipine Besylate and Olmesartan Medoxomil in Bulk Drug and Formulation

Two methods are described for simultaneous determination of amlodipine besylate and olmesartan medoxomil in formulation. The first method was based on the HPTLC separation of two drugs on Merck HPTLC aluminium sheets of silica gel 60 F₂₅₄ using n-butanol: acetic acid: water (5:1:0.1, v/v/v) as the mobile phase. The second method was based on the HPLC separation of the two drugs on the RP-PerfectSil-100 ODS-3–C₁₈ column from MZ-Analysetechnik GmbH, Germany and acetonitrile/0.03 M ammonium acetate buffer (pH = 3) in a ratio of 55:45 as the mobile phase. Both methods have been applied to formulation without interference of excipients of formulation.     

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.     

The hydrophilic interaction like properties of some reversed phase high performance liquid chromatography columns in the analysis of basic compounds

The hydrophilic interaction chromatography (HILIC) like properties of an ACE cyano (CN) HPLC column was studied for the separation of some basic compounds. Good separation of a test mix of basic compounds was obtained with a mobile phase consisting of acetonitrile/water (95:5) containing 3.25 mM ammonium acetate. The retention times of the basic compounds decreased with increased ionic strength or with increased water content in the mobile phase. When Trishydroxymethyl aminomethane (Tris) (pK(a) 8.0), which is a weaker amine than ammonia (pK(a) 9.3), was used as an additive in the mobile phase retention of the basic compounds was increased. The ACE CN column gave excellent peak shapes for all the basic compounds. The utility of the column for impurity profiling of two basic drugs was tested and some impurities in oxprenolol were characterised by interfacing with Fourier transform mass spectrometry. It was also observed that ACE butyl and ACE phenyl columns retained basic compounds when the columns were eluted with a mobile phase consisting of acetonitrile/water (95:5) containing 3.25 mM ammonium acetate.     

液相色谱-串联质谱法快速筛查马饲料中39种违禁药物

建立了同时快速筛查和确证马饲料中39种赛马违禁药物(包含抗心率失常类药物、抗惊厥类药物、止痉挛类药物、抗疟疾类药物、刺激剂、麻醉剂及大麻酚类药物)的液相色谱串联质谱(HPLC-MS/MS)分析方法。样品粉碎后分别经1 mmol/L HClO4溶液和酸化乙腈溶液提取,并通过混合型固相萃取柱净化。采用Agilent Zorbax SB(10.0 cm×2.1 mm i.d.,3.5 μm) 色谱柱,以0.4%甲酸水溶液和0.4%甲酸乙腈为流动相进行梯度洗脱,正离子多反应监测(MRM) 模式下检测。方法对空白饲料3个加标水平下的平均回收率为58%~116%,相对标准偏差为1.6%~20.4%,各类药物线性良好(r2＞0.99)。方法检出限(S/N≥3)和定量下限(S/N≥10)分别为0.2~25.0 μg/kg和1.0~40.0 μg/kg,其中超过67%药物的检出限在2.5 μg/kg以下。实验结果表明,该方法分析时间短,灵敏度、精密度良好,适用于马饲料中上述几类药物的快速筛查和测定。