Development of HPLC-MS/MS method for the simultaneous determination of metabolites of organophosphate pesticides,synthetic pyrethroids,herbicides and DEET in human urine

We developed an isotopic dilution high-performance liquid chromatography (HPLC)/tandem mass spectrometer (MS/MS) method to rapidly and accurately quantify nine metabolites of several classes of pesticide in 1 mL human urine specimens. The analytes covered in the method are two organophosphate (OP) pesticide metabolites: 3,5,6-trichloro-2-pyridinol (TCPy), 2-isopropyl-6-methyl-4-pyrimidinol (IMPY); three synthetic pyrethroid metabolites: 3-phenoxy benzoic acid (3-PBA), 4-fluoro-3-phenoxybenzoic acid (4-F-3-PBA) and trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-1(1-cyclopropane) carboxylic acid (t-DCCA); three herbicide metabolites: 2,4-dichlorophenoxyacetic acid (DCPAA), 2,4,5-trichlorophenoxyacetic acid (TCPAA) and atrazine mercapturate; and one insect repellent: N,N-diethyl-meta-toluamide (DEET). The analytes are first deconjugated by incubating with acetate/β-glucuronidase buffer at 37°C for 17 h. The deconjugated analytes are extracted and concentrated from the urine matrix using solid-phase extraction cartridges, separated through C18 reversed phase HPLC, and analysed on MS/MS. The MS/MS was operated in positive and negative electrospray ionisation switch mode. Two ions from each analyte and one from each labelled internal standard are monitored for quantification and confirmation. The limit of detections (LODs) for all the analytes are in the low parts-per-trillion (0.05 ng/mL) except TCPy where it was 0.5 ng/mL) with a wide linear range (0.05 up to 40 ng/mL) and provides high accuracy (recoveries: 90–118%) and high precision (coefficient of variation <15%). The method accuracy was also verified by the analysis of proficiency testing urine samples. We analysed 101 urine samples for a recent California study cohort, and detection frequencies ranged from ~100% to 0%: 3-PBA (98%), IMPY (91%), TCPy, (89%), DCPAA (66%), 4-F-3-PBA (11%), TCPAA (0%).     

A fully validated isotope dilution HPLC-MS/MS method for the simultaneous determination of succinylcholine and succinylmonocholine in serum and urine samples

A high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the simultaneous detection of succinylcholine (SUX) and its metabolite succinylmonocholine (SMC) in serum and urine is presented. For internal standardization using isotope dilution, the deuterated compounds SUX-d(18) and SMC-d(3) were employed. Full validation was performed according to international guidelines. Solid-phase extraction (SPE) of acidified samples was accomplished using Strata-X polymeric reversed phase cartridges together with heptafluorobutyric acid (HFBA) as ion-pairing reagent. Separation was achieved within 13 min on a Phenomenex Synergi Hydro RP C18 column (4 microm, 150 x 2 mm) using a gradient of 5 mM ammonium formate buffer pH 3.5 and acetonitrile.To ensure the method's applicability in forensic as well as clinical toxicology, the specific demands of both research fields were taken into account, and the method was thus validated for a low and high concentration range. For both serum and urine as sample matrix, the validation revealed good intraday and interday precisions, consistently ranging below 15% for the lowest and below 10% for elevated concentrations. Accuracy was likewise good and never exceeded 10%. Extraction recovery was excellent, ranging between 88.1 and 103.9% for SUX and SMC in both tested matrices. Matrix effects were significant, the otherwise optimized extraction and detection methods, however, allowed for a very satisfactory sensitivity of the described method: For serum, the limits of detection and quantitation were determined to be 1.9 and 6.0 ng/ml for SUX, as well as 2.5 and 8.6 ng/ml for SMC, respectively; for urine, the corresponding values were established to be 1.4 and 4.0 ng/ml (SUX), as well as 1.5 and 4.9 ng/ml (SMC).The presented method was successfully applied to authentic samples of two forensic cases investigated in the institute of forensic medicine in Bonn, allowing the diagnosis of SUX intoxications.     

高效液相色谱-串联质谱法测定猪尿中秋水仙碱

提出了猪尿中秋水仙碱的液相色谱-串联质谱检测方法。采用pH8.0磷酸盐缓冲溶液碱化5mL尿样,用乙酸乙酯提取两次,合并提取液,于40℃吹氮蒸干,用2 mL流动相溶解残渣。在色谱分离中选用Sunfire C_(18)色谱柱,流动相为甲醇-2mmol·L~(-1)乙酸铵溶液(含0.1%甲酸)混合溶液(40+60),流量为0.2mL·min,进样量为10μL。质谱测定中采用电喷雾正离子模式离子化、多反应监测模式测定秋水仙碱,外标法定量。猪尿中秋水仙碱在1～50μg·L~(-1)内呈线性,测定下限(10S/N)为0.2μg·L~(-1),方法的回收率为90%～100%,相对标准偏差(n=6)小于9.0%。     

固相萃取地表水中痕量联苯胺及HPLC-MS测定

采用固相萃取及高效液相色谱-串联质谱技术,建立了地表水中痕量联苯胺的测定方法.水样经HLB固相萃取柱富集,二氯甲烷与丙酮(1:1,v/v)洗脱,氮吹后转为甲醇溶剂,以液相色谱串联质谱选择离子监测(SRM)模式定性、定量分析.在本实验条件下,加标回收率在72.0%～94.0%之间,相对标准偏差8.1%～9.8%(n=7)...     

Development,validation, and implementation of an UHPLC–MS/MS method for the quantitation of furosemide in infant urine samples

Furosemide is a diuretic drug used to increase urine flow in order to reduce the amount of salt and water in the body. It is commonly utilized to treat preterm infants with chronic lung disease of prematurity. There is a need for a simple and reliable quantitation of furosemide in human urine. We have developed and validated an ultra-high performance liquid chromatography–tandem mass spectrometry method for furosemide quantitation in human urine with an assay range of 0.100–50.0 μg/ml. Sample preparation involved solid-phase extraction with 10 μl of urine. Intra-day accuracies and precisions for the quality control samples were 94.5–106 and 1.86–10.2%, respectively, while inter-day accuracies and precision were 99.2–102 and 3.38–7.41%, respectively. Recovery for furosemide had an average of 23.8%, with an average matrix effect of 101%. Furosemide was stable in human urine under the assay conditions. Stability for furosemide was shown at 1 week (room temperature, 4, −20 and −78°C), 6 months (−78°C), and through three freeze–thaw cycles. This robust assay demonstrates accurate and precise quantitation of furosemide in a small volume (10 μl) of human urine. It is currently being implemented in an ongoing pediatric clinical study.     

Automated on-line column-switching HPLC-MS/MS method for measuring environmental phenols and parabens in serum

We developed a method using on-line solid phase extraction (SPE) coupled to high performance liquid chromatography–isotope dilution tandem mass spectrometry (HPLC–MS/MS) to measure the serum concentrations of seven environmental phenols and five parabens: bisphenol A; ortho-phenylphenol; 2,4-dichlorophenol; 2,5-dichlorophenol; 2,4,5-trichlorophenol; benzophenone-3; triclosan; and methyl-, ethyl-, propyl-, butyl-, and benzyl-parabens. The phenols and parabens present in serum were retained and concentrated on a C18 reversed-phase size-exclusion SPE column, back-eluted from the SPE column while the eluate was diluted through a mixing Tee (analyte peak focusing), separated using a pair of monolithic HPLC columns, and detected by isotope dilution-MS/MS. Sample preparation did not require protein precipitation, only dilution of the serum with 0.1 M formic acid. This method, which combines an on-line SPE with analyte peak focusing feature and the selective atmospheric pressure photoionization MS detection, resulted in limits of detection ranging from 0.1 to 0.5 ng/mL for most of the analytes. The high throughput and adequate sensitivity with yet a relative low serum volume used (100 μL) confirm that analytically it is possible to measure simultaneously these phenols and parabens with the precision and accuracy at sub-parts-per-billion levels required for biomonitoring. However, important additional factors, including validated sample collecting, handling, and storing protocols, as well as toxicokinetic data, are required if these measures are used for exposure assessment.     

高通量固相萃取-超高效液相色谱-串联质谱法测定人尿中8种环境酚类内分泌干扰物

建立了人尿中8种环境酚类化合物的96孔板固相萃取-超高效液相色谱-串联质谱(96-well SPE LC-MS/MS)检测方法,其中包括7种双酚类化合物和三氯生。尿样解冻到室温,经β-葡萄糖醛酸苷肽酶/芳基磺酸酯酶37℃过夜酶解。实验比较了3种96孔板固相萃取柱和不同淋洗条件对人尿样的净化效果和目标化合物的回收率。结果显示,采用Oasis HLB 96孔板(60 mg)对样品进行萃取和用30%(v/v)乙腈水溶液进行淋洗净化的纯化效果最好。纯化后目标物用甲醇溶液洗脱,经氮气吹干,用0.5 mL甲醇-水(1∶1, v/v)溶液定容,目标化合物用UPLC-MS/MS进行检测。比较了2种分析柱(C₁₈和T3分析柱)以及不同的有机流动相对分离样品中目标物的影响。结果显示,以BEH C₁₈(100 mm×2.1 mm, 1.7μm)作为分析柱,乙腈/水作为流动相,以流速0.3 mL/min梯度洗脱时,目标物的分离效果最好。质谱条件选择串联质谱负离子电喷雾(ESI^-)多反应监测模式(MRM)进行检测。对样品的基质效应进行评估发现,双...     

田七花提取物中22种皂苷类成分的液相色谱-质谱测定

在甲酸体系中以高效液相色谱负离子模式电喷雾电离质谱以及碰撞诱导裂解技术研究了12种人参皂苷(Re、Rg1、Rg2、Rg3、Rf、Rb1、Rb2、Rb3、Rc、Rd、Rh1 和Rh2).结果表明,应用皂苷化合物(包括人参皂苷、田七皂苷和绞股蓝皂苷)的质谱及裂解规律可在缺少相应对照品的情况下对其进行可靠的鉴定.在此基础上,对田七花样品以加压溶剂萃取法提取,然后以LC-MS/MS分析,从中鉴定出22种皂苷,其中六糖皂苷Ⅰ和Ⅱ、乙酰基Rb1为首次报道,并且定量测定了其中10种皂苷的含量.     

Rapid and simultaneous determination of multiple classes of abused drugs and metabolites in human urine by a robust LC‐MS/MS method – application to urine drug testing in pain clinics

A simple LC‐MS/MS method was developed and validated for quantitatively analyzing six classes of 26 abused drugs and metabolites in human urine: (1) illicit drugs; (2) opiates; (3) synthetic opioids; (4) sedative; (5) stimulants; and (6) γ‐aminobutyric acid analogs. All urine samples were diluted with a mixture of isotope‐labeled internal standards, hydrolyzed with β‐glucuronidase and directly injected in a gradient chromatographic run. The mobile phase was composed of 0.1% formic acid in water and 0.1% of formic acid in methanol. A 4.9 min run time using the multiplexing driver and ultra‐biphenyl column (50 × 2.1 mm, 5 µm, RESTEK) allowed all drugs to have sufficient resolution in a short elute time. The overlapping liquid chromatography runs and scheduled multiple reaction monitoring acquisition method resulted in a higher overall throughput for the system. The result was linear over the studied range (2–16,000 ng/mL) for all compounds with correlation coefficients r² ≥ 0.995. The intra‐day and inter‐day precisions and accuracies were within 15% and recovery was between 83 and 115% for all analytes. Freeze–thaw stability for three cycles and long‐term stability (57 days, ?20°C) were established for all analytes. The cross‐validation between College of American Pathologists and in‐house was validated (0.06% ≤ bias ≤ 12.3%). The applicability of the method was examined by analyzing urine samples from chronic pain patients (n = 610). Copyright © 2013 John Wiley & Sons, Ltd.     

高效液相色谱-串联质谱法测定生鲜乳中三聚氰胺的含量

采用高效液相色谱-串联质谱法测定生鲜乳中三聚氰胺残留量。样品经乙腈溶液超声提取,过Waters Oasis MCX固相萃取小柱净化,50℃氮气吹干,再用1.0mL乙腈溶解后供高效液相色谱-串联质谱分析。以Waters Hilic色谱柱(100 mm×2.1 mm,3μm)为固定相,用乙腈-5mmol·L-1乙酸铵(95+5)溶液洗脱,采用电喷雾正离子模式多反应监测,内标法定量。三聚氰胺的质量浓度在10.0μg·L-1以内呈线性,检出限(3S/N)为0.5μg·kg-1。取空白样品在3个标准加入水平下进行回收和精密度试验,回收率在99.8%~103%之间,测定值的相对标准偏差(n=10)在1.9%~3.2%之间。     

Development and validation of an LC–MS/MS Method for the quantitation of heparan sulfate in human urine

Heparan sulfate is a linear polysaccharide and serves as an important biomarker to monitor patient response to therapies for MPS III disorder. It is challenging to analyze heparan sulfate intact owing to its complexity and heterogeneity. Therefore, a sensitive, robust and validated LC–MS/MS method is needed to support the clinical studies for the quantitation of heparan sulfate in biofluids under regulated settings. Presented in this work are the results of the development and validation of an LC–MS/MS method for the quantitation of heparan sulfate in human urine using selected high‐abundant disaccharides as surrogates. During sample processing, a combination of analytical technologies have been employed, including rapid digestion, filtration, solid‐phase extraction and chemical derivatization. The validated method is highly sensitive and is able to analyze heparan sulfate in urine samples from healthy donors. Disaccharide constitution analysis in urine samples from 25 healthy donors was performed using the assay and demonstrated the proof of concept of using selected disaccharides as a surrogate for validation and quantitation.     

高效液相色谱-串联质谱法检测生物样品中6种雌激素

建立了雌酮、雌二醇、雌三醇、己烯雌酚、己烷雌酚和炔雌醇6种雌激素在生物体中的HPLC-MS/MS分析方法.采用加速溶剂萃取、固相萃取技术进行提取、富集及净化,有效降低了基质的干扰.以甲醇-0.1％氨水溶液为流动相,以C18色谱柱进行分离,质谱采用电喷雾负离子扫描模式,6种雌激素的回收率为88％～104％,相对标准偏差在1.3％～8.3％之间.雌酮、雌二醇、雌三醇在生物体中的方法检出限0.35ng/g;己烯雌酚、己烷雌酚、17α-乙炔基雌二醇在生物体中的方法检出限为0.13ng/g.方法适用于生物体内雌激素的分析和检测.     

Development of LC‐MS/MS method for the determination of dapiprazole on dried blood spots and urine: application to pharmacokinetics

A rapid and highly sensitive liquid chromatography–tandem mass spectrometric (LC‐MS/MS) method for determination of dapiprazole on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse‐phase C₁₈ column (250 × 4.6 mm i.d., 5 µm), using 20 mm ammonium acetate (pH adjusted to 4.0 with acetic acid) and acetonitrile (80:20, v/v) as a mobile phase at 25 °C. LC‐MS detection was performed with selective ion monitoring using target ions at m/z 326 and m/z 306 for dapiprazole and mepiprazole used as internal standard, respectively. The calibration curve showed a good linearity in the concentration range of 1–3000 ng/mL. The effect of hematocrit on extraction of dapiprazole from DBS was evaluated. The mean recoveries of dapiprazole from DBS and urine were 93.88 and 90.29% respectively. The intra‐ and inter‐day precisions were <4.19% in DBS as well as urine. The limits of detection and quantification were 0.30 and 1.10 ng/mL in DBS and 0.45 and 1.50 ng/mL in urine samples, respectively. The method was validated as per US Food and Drug Administration guidelines and successfully applied to a pharmacokinetic study of dapiprazole in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Validation of a sensitive LC/MS/MS method for the determination of zidovudine and lamivudine in human plasma

A sensitive LC/MS/MS assay for determining zidovudine (ZDV) and lamivudine (3TC) in human plasma was validated to support antiretroviral pharmacology research programs. After addition of stable labeled isotopic zidovudine (ZDV‐IS) and lamivudine (3TC‐IS) as internal standard, a solid‐phase extraction was performed with an Oasis HLB 1 cm³ cartridge, with recoveries of 92.3% for ZDV and 93.9% for 3TC. A Phenomonex Synergi Hydro‐RP (2.0 × 150 mm) reversed‐phase analytical column was utilized for chromatographic separation. The mobile phase consisted of an aqueous solution of 15% acetonitrile and 0.1% acetic acid. Detection was accomplished by ESI/MS/MS in the positive ion mode, monitoring 268/127, 271/130, 230/112 and 233/115 transitions, for ZDV, ZDV‐IS, 3TC and 3TC‐IS, respectively. The method was linear from 1 to 3000 ng/mL with a minimum quantifiable limit of 1 ng/mL when 100 μL of plasma was analyzed. Validation results demonstrated high accuracy (≤8.3% deviation) and high precision (≤10% CV) for the quality control samples. The method was also shown to be specific and reproducible. The value of the high sensitivity was demonstrated by quantitation of approximately 100 existing samples that had ZDV below the limit of quantitation using a previously validated, less sensitive HPLC‐UV method utilized in the laboratory. Copyright © 2011 John Wiley & Sons, Ltd.     

Rapid and sensitive HPLC-MS/MS method for pharmacokinetic assessment of ribavirin in healthy Chinese

A rapid and sensitive quantitative assay method was developed for determining ribavirin pharmacokinetic in human plasma. The chromatographic separation was achieved within 4.5 min using a SinoChrom ODS-BP column (4.6 x 150 mm, 5 microm) with acetonitrile-water (1 mmol/L ammonium acetate buffer, 0.1% formic acid; 15:85, v/v) at a constant flow rate of 0.8 mL/min. The MRM pairs were m/z 245.2 --> m/z 113.1 for ribavirin and m/z 226.1 --> m/z 152.1 for acyclovir (internal standard), respectively, with dwell times of 200 ms for each transition. The results showed calibration curve for ribavirin was linear over a concentration range of 1-1000 ng/mL. The lower limit of quantification (LLOQ) was 1 ng/mL ribavirin. Twenty healthy volunteers received a 300 mg oral dose of ribavirin. Blood samples were then collected up to 120 h postdosing. All plasma data were comodeled for ribavirin by using noncompartmental modeling. The single dose of ribavirin was well tolerated and no serious adverse effects occurred. The mean time to maximum concentration was about 1.25 h. The mean maximum concentration of drug in plasma for oral ribavirin was 250 ng/mL. The mean elimination half-life was 43.6 h. The present study describes a simple, specific, sensitive HPLC-MS/MS method for measuring plasma drug concentration and analyzing human pharmacokinetics of ribavirin.     

高效液相色谱-串联质谱法测定农产品中矮壮素残留量

提出了农产品中矮壮素的高效液相色谱-串联质谱(HPLC-MS/MS)分析方法。样品经甲醇-水(1+1)溶液提取,正己烷液液萃取后,采用阳离子交换固相萃取柱净化。所得净化液以阳离子交换树脂与C18混合填料色谱柱为固定相,以含0.1%(体积分数)乙酸的乙腈-10mmol·L-1乙酸铵(1+1)溶液为流动相进行等度洗脱,采用电喷雾正离子源,多反应监测模式检测,同位素内标法定量。矮壮素的线性范围为1.0~100μg·L-1,检出限(3S/N)为5μg·kg-1,测定下限(10S/N)为10μg·kg-1。矮壮素在10,100,500μg·kg-1等3个加标水平的回收率为90.5%~98.5%之间,测定值的相对标准偏差(n=6)在0.99%~2.2%之间。     

高效液相色谱-质谱/质谱测定血浆中格列苯脲

本文建立了人血浆中格列苯脲的高效液相色谱-质谱/质谱(HPLC-MS/MS)分析方法。血浆中格列苯脲测定的线性范围是1~300 ng/mL;高、中、低三个浓度分析测定的精密度(RSD)<15%;格列苯脲和格列齐特(内标)绝对回收率均>80%。本方法灵敏、准确,重现性好,用于血药浓度的测定获得满意结果。     

Cryogenic grinding pre-treatment improves extraction efficiency of fluoroquinolones for HPLC-MS/MS determination in animal tissue

An efficiency extraction of fluoroquinolones in chicken muscle was achieved by pulverizing it in a freezer mill before treatment with NaOH (10mM)/MeCN (1:1). The improvement of cryogenic grinding in the extraction was demonstrated for the same piece (whole leg) of four chickens treated with enrofloxacin in equal doses. A confirmatory method based on high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was used to analyze the extracts. The chromatographic separation was achieved in 5min with a Synergi Fusion-RP 80A (50 x 2mm, 4μm) column filled with a hybrid polymer. The HPLC was coupled with a detector based in a quadrupole–linear ion trap Q-TRAP that allows a confirmatory detection according to the European legislation. The specificity of the method was assessed by testing a number of representative blank muscle samples (n = 10) to verify the absence of potential interfering compounds. The limits of detection and quantitation were 2 and 5ng g^-1 of quinolones in muscle samples, respectively. The chromatographic method was demonstrated to be linear for the range studied (5–500ng g^-1) with the P value for lack-of-fit in the ANOVA table greater or equal to 0.10 (calibration coefficient 0.9998 and 0.9996 for ciprofloxacin and enrofloxacin, respectively). The mean intra-day relative standard deviation (RSD) (n = 6, c = 50ng g^-1) was 6%; inter-day assay gave a RSD of 12%. The extraction and clean-up were carried out in one step with very satisfactory recovery data (between 65 and 101%).     

Developing LC–MS/MS methods to quantify rivaroxabanin human plasma and urine: Application to therapeutic drug monitoring

Rivaroxaban is an oral anticoagulant directly inhibiting the activity of Factor Xa, which is widely used for the prophylaxis of thromboembolic disorders. Therapeutic drug monitoring (TDM) is required during therapy for individual dosage adjustment. This study aimed at developing a liquid chromatography/tandem mass spectrometry method that was suitable for rivaroxaban TDM in human plasma and urine and exploring the feasibility of urine drug monitoring in medical care. A 3 min run time of the LC–MS/MS methods was established by employing an Acquity UPLC BEH C₁₈ (2.1 × 50 mm, 1.7 μm) column using gradient elution of 10 mmol/L ammonium acetate containing 0.1% formic acid–0.1% formic acid acetonitrile as a mobile phase at a flow rate of 0.4 ml/min with calibration ranges of 0.5–400 and 10–10,000 ng/ml for human plasma and urine, respectively. Rivaroxaban was detected on a triple quadrupole tandem mass spectrometer with an electrospray ionization source in positive ion mode. The methods showed good linearity within the calibration range. The precision and accuracy, matrix effect, extraction recovery and stability in both human matrices were all validated and meet the international guideline requirements. These validated methods were successfully applied to support the TDM of an aged patient receiving rivaroxaban for therapy.     

Dispersive solid‐phase extraction procedure coupled to UPLC‐ESI‐MS/MS analysis for the simultaneous determination of thirteen cytotoxic drugs in human urine

A fast and easy tailored dispersive solid‐phase extraction (d‐SPE) procedure has been developed for the determination of 13 cytostatic drugs. Combined with a rapid and simultaneous ultra performance liquid chromatography/tandem mass spectrometry method for residue identification and quantification in urine, it has been fully validated and tested to study a realistic situation in working environment. The target compounds were chosen from the most common classes used in hospitals. The d‐SPE adsorbent was obtained mixing Oasis HLB® with C₁₈ and applied to a large volume of sample (10 mL). The electrospray ionization‐mass spectrometry acquisition was conducted in a mixed period mode: six acquisition windows were in positive ionization and one in negative (for 5‐fluorouracil). The lowest limit of quantification was found at 0.04 μg/L urine for methotrexate. The absolute recovery of cytotoxic drugs was assessed at two concentrations levels and ranged from 67.1% (cytarabine) to 102.3% (etoposide) and from 65.3% (cytarabine) to 101.2% (methotrexate) for the lower and higher levels, respectively, with the relative standard deviation always <12%. This method gives the opportunity to analyze drugs in a wide molecular weight range (from 130 to 853 a.m.u.) and in a complex matrix, such as urine, without losing any of the features that a method intended for trace quantification must have. Copyright © 2016 John Wiley & Sons, Ltd.