Simultaneous analysis of delta9-THC and its major metabolites in urine, plasma, and meconium by GC-MS using an immunoaffinity extraction procedure

A simple extraction procedure for delta9-tetrahydrocannabinol (delta9-THC) and its metabolites from various biological specimens was developed based on immunoaffinity chromatography. Using the affinity resin prepared by immobilization of THC antibody onto cyanogen bromide-activated Sepharose 4B, delta9-THC and its major metabolites including 11-nor-delta9-THC-9-carboxylic acid (delta9-THCCOOH), 11-hydroxy-delta9-THC (11-OH-delta9-THC), and 8beta,11-dihydroxy-delta9-THC (8beta,11-diOH-delta9-THC) were extracted simultaneously from plasma or urine after enzyme hydrolysis. The samples were derivatized as TMS derivatives and analyzed by gas chromatography-mass spectrometry in EI mode with SIM monitoring. Greater than 87% extraction recovery of the four analytes was obtained from both plasma and urine at 5 and 50 ng/mL concentration levels. The method was also used for meconium analysis with some modification. The extraction recovery from meconium, however, was lower than that of plasma and urine, ranging from 52 to 72% at the 10-ng/g level. All compounds showed good linearity within the tested ranges up to 100 ng/mL (g). The limits of detection ranged from 0.5 to 2.5 ng/mL in plasma and urine, and from 1.0 to 2.5 ng/g in meconium. Analysis of 24 meconium specimens showed that 11-OH-delta9-THC is indeed an important metabolite in meconium.     

Simultaneous determination of codeine, morphine, hydrocodone, hydromorphone, oxycodone, and 6-acetylmorphine in urine, serum, plasma, whole blood, and meconium by LC-MS-MS

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for simultaneous analysis of six major opiates in urine, serum, plasma, whole blood, and meconium is described. The six opiates included are codeine, morphine, hydrocodone, hydromorphone, oxycodone, and 6-acetylmorphine (6-AM). The method was compared to an in-house gas chromatography (GC)-MS method and an LC-MS-MS method performed by another laboratory. The sample preparation time was decreased by eliminating the glucuronide hydrolysis and derivatization required for GC-MS analysis, as well as by adapting the solid-phase extraction to elute directly into autosampler vials. These improvements illustrate the advantages of an LC-MS-MS method over a GC-MS method for opiates. The structural similarity of these six opiates and others in the opiate class causes a high potential for interference and false-positive results. Twelve opiate analogues and metabolites were evaluated for interference. The potential for interference was reduced by altering the MRM transitions chosen for the six opiates. The increased specificity of LC-MS-MS decreased the interference rate in urine to 3.9% compared to 13.6% on the in-house GC-MS method. The rate of positivity for 6-AM in meconium is described for the first time. In urine, 11.0% of morphine positive specimens were also positive for 6-AM compared to 8.3% in serum/plasma and 0.9% in meconium. Although 6-AM is infrequent in meconium, it provides a definitive proof of illegal heroin abuse by the pregnant mother. This method has been routinely used in our laboratory over the last 6 months on more than 1500 patient specimens.     

Quantification of imatinib in human plasma by high-performance liquid chromatography-tandem mass spectrometry

Imatinib, also known as Gleevec or Glivec, is a selective tyrosine kinase inhibitor currently used for the treatment of Philadelphia chromosome-positive chronic myeloid leukemia (CML) and for other malignant pathologies. We have developed a LC-MS-MS [corrected] method that could be used for imatinib therapeutic drug monitoring in plasma. After a liquid-liquid extraction, the imatinib and its deuterated internal standard were eluted on an XTerra RP18 column with a gradient of acetonitrile-ammonium formiate buffer 4 mmol/L, pH 3.2. Imatinib was detected by electrospray ionization mass spectrometry with multiple reaction-monitoring mode. The calibration curves were linear over the range 10-5000 ng/mL. The limit of quantification was set at 10 ng/mL. The bias was lower than 8%. Intra-day and inter-day precisions were lower than 8%. The extraction recovery was higher than 90%. This method is simple, adapted to routine application, and allows accurate therapeutic monitoring of imatinib. It can be used to evaluate patient adherence to daily oral therapy, drug-drug interactions, or pharmacokinetic/pharmacodynamic relationships.     

Improved Detection of Opioid Use in Chronic Pain Patients through Monitoring of Opioid Glucuronides in Urine

When chronic pain patients are suspected of being non-compliant, their therapy can be withdrawn. Therefore, sensitive and specific confirmatory testing is important for identifying diversion and adherence. This work aimed to develop a novel liquid chromatography tandem mass spectrometry (LC-MS-MS) method to detect 14 opioids and six opioid glucuronide metabolites in urine with minimal sample preparation. Analytes included were morphine, oxymorphone, hydromorphone, oxycodone, hydrocodone, codeine, fentanyl, norfentanyl, 6-monoacetylmorphine, meperidine, normeperidine, propoxyphene, methadone, buprenorphine, morphine-3-glucuronide, morphine-6-glucuronide, oxymorphone glucuronide, hydromorphone glucuronide, codeine-6-glucuronide and norbuprenorphine glucuronide. Samples were processed by centrifugation and diluted in equal volume with a deuterated internal standard containing 14 opioids and four opioid glucuronides. The separation of all compounds was complete in nine minutes. The assay was linear between 10 and 1,000 ng/mL (fentanyl 0.25-25 ng/mL). Intra-assay imprecision (500 ng/mL, fentanyl 12.5 ng/mL) ranged from 1.0 to 8.4% coefficient of variation. Inter-assay precision ranged from 2.9 to 6.0%. Recovery was determined by spiking five patient specimens with opioid and opioid glucuronide standards at 100 ng/mL (fentanyl 2.5 ng/mL). Recoveries ranged from 82 to 107% (median 98.9%). The method correlated with our current quantitative LC-MS-MS assay for opioids, which employs different chromatography. Internal standards were not available for every analyte to critically evaluate for ion suppression. Instead, a novel approach was designed to achieve the most rigorous quality control possible, in which the recovery of each analyte was evaluated in each negative sample.     

Direct analysis of opiates in urine by liquid chromatography-tandem mass spectrometry

A method for the direct analysis of 10 opiate compounds in urine was developed using liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) with electrospray ionization interface (ESI). Opiates included were morphine-3-P-glucuronide, morphine-6--glucuronide, morphine, oxymorphone, hydromorphone, norcodeine, codeine, oxycodone, 6-monoacetylmorphine (6MAM), and hydrocodone. Urine samples were prepared by centrifugation to remove large particles and direct injection into the LC-MS-MS. Separation and detection of all compounds was accomplished within 6 min. Linearity was established for all opiates except 6MAM from 50 ng/mL to 10,000 ng/mL; 6MAM from 0.25 ng/mL to 50 ng/mL with all correlation coefficients (r) > 0.99. Interrun precision (%CV) ranged from 1.1% to 16.7%, and intrarun precision ranged from 1.3% to 16.3%. Accuracy (% bias) ranged from -7.3% to 13.6% and -8.5% to 11.8 for inter- and intrarun, respectively. Eighty-nine urine samples previously analyzed by gas chromatography-MS were re-analyzed by the LC-MS-MS method. The qualitative results found an 88% agreement for negative samples between the two methods and 94% for positive samples. The LC-MS-MS method identified 19 samples with additional opiates in the positive samples. Overall, the direct injection LC-MS-MS method performed well and permitted the rapid analysis of urine samples for several opiates simultaneously without extensive sample preparation.     

Simultaneous analysis of frequently used licit and illicit psychoactive drugs in breast milk by liquid chromatography tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC-MS-MS) method for the quantification of frequently used licit (caffeine, nicotine and cotinine) and illicit drugs (opiates, cocaine, cannabinoids and amphetamines) in breast milk was developed and fully validated. Chromatography was performed on a reverse-phase column using a gradient of 2mM ammonium acetate, pH 6.6, and methyl alcohol as mobile phase at a flow rate of 0.35 mL/min. Separated analytes were quantified by electrospray ionization tandem mass spectrometry in positive ion mode using multiple reaction monitoring. Milk samples were kept at -20 °C until analysis and the compounds under investigation were extracted from the matrix by Bond Elut Certify cartridges. The concentration range covered was LOQ to 1000 ng/mL for all the investigated drugs. Intra- and inter-assay imprecision was less than 20%, analytical recovery ranged between 51.6% and 86.5%, matrix effect between 71.1% and 116.6% and process efficiency between 46.8% and 84.0%. Analytes were stable after three freeze-thaw cycles, after 6 months at -20 °C and after the pasteurization process (differences to the initial concentration always lower than 10%). matrix effect ranged from 77.6% to 116.6%, recovery from 51.6% to 86.5%, and process efficiency from 46.8% to 79.0%. This LC-MS-MS assay was applied to screen samples from the largest Spanish milk bank and samples coming from drug addicted mothers. The developed method provided adequate sensitivity and performance characteristics to prove the presence of only caffeine in a small percentage of samples from milk donating nursing mothers and the presence or absence of most commonly used illicit drugs in breast milk from addicted lactating mothers.     

Determination of tapentadol and its metabolite N-desmethyltapentadol in urine and oral fluid using liquid chromatography with tandem mass spectral detection

An analytical procedure for the determination of the new pain medication tapentadol and its main metabolite N-desmethyltapentadol (DMT), in urine and oral fluid has been developed and validated using liquid chromatography with tandem mass spectral detection (LC-MS-MS). Oral fluid was collected using Quantisal? devices, and drugs present were quantified using solid-phase extraction followed by LC-MS-MS. For confirmation, two transitions were monitored and one ratio determined which had to be within 20% of that of the known calibration standard. For tapentadol, 222.1 > 107 was used as the quantifying transition; 222.1 > 121 for the qualifier. For DMT, 208.1 > 107 was used for quantification; 208.1 > 121 as the qualifier. For saliva, the linear range was 10-100 ng/mL; the lower limit of quantitation (LLOQ) was 10 ng/mL; the intraday precision was 3.6% (n = 6) and interday precision was 13.6% (n = 24). The recovery of tapentadol and DMT from the oral fluid collection pad was > 99%. For urine, the specimens were diluted and injected directly into the LC-MS-MS. The LLOQ was 50 ng/mL; the intraday and interday precisions were 2.1% and 4.4%, respectively, for tapentadol and 2.9% and 5.7%, respectively, for DMT. This is the first analytical procedure for tapentadol and DMT in urine and oral fluid.     

A rapid and sensitive technique for assessing exposure to VX via GC-MS-MS analysis

A rapid and sensitive method for the determination of the chemical warfare agent VX in plasma taken from G?ttingen minipigs has been developed using isotope-dilution gas chromatography-tandem mass spectrometry (GC-MS-MS). Chromatographic separation was achieved on a 5% diphenyl/95% dimethyl polysiloxane capillary column with a total run time of about 11 min. The analyte was detected using ammonia chemical ionization in the multiple reaction monitoring mode, following a simple extraction with 10% 2-propanol in hexane. A good linear relationship was obtained in the quantitative concentration range of 10 ng/mL to 1000 ng/mL (r(2) = 0.9998) with an average slope of 1.275 +/- 0.037 (n = 7), and an absolute detection limit of 0.4 pg on column. The average recovery for VX was 95% in saline in the concentration range of 50-100 ng/mL. The method was successfully applied to the analysis of VX in minipig plasma in a preliminary toxicokinetic study.     

HPLC—MS／MS测定人体血浆和尿液中帕洛诺司琼的浓度

目的建立一种简便、灵敏的测定人体血浆和尿液中帕洛诺司琼浓度的高效液相色谱一串联质谱（HPLC-MS／MS）方法。方法血浆、尿液样品分别采用甲醇沉淀处理后,选样分析。采用Agilent-ZORBAX-C18色谱柱（2．1mm×50mm,5fμm）．以乙腈-0．1％甲酸溶液为流动相,采用正离子,多反应监测方式测定样品浓度。用于定量分析的检测离子质荷电（m／z）297．2→m/z110．1（帕洛诺司琼）和DI／Z285．0→M／z193．0（内标）。结果帕洛诺司琼血浆样品在0．02～10ng·mL^-1与峰面积线性关系良好（r=0．9975）;定量下限（LLOQ）为0．02ng·L^-1;日内与日间RSD均〈10％;回收率在89．6％～114．0％。尿样在2．5～100ng·mL^-1与峰面积线性关系良好,7—0．9974;定量下限（LLOQ）为2．5ng·mL^-1;日内与日间RSD均〈10％;回收率在96．4％～113．4％。结论本方法简便快速、灵敏准确,适用于帕洛诺司琼在人体体内的药物动力学研究。     

Simultaneous determination of albendazole metabolites, praziquantel and its metabolite in plasma by high-performance liquid chromatography-electrospray mass spectrometry

The analysis of albendazole sulfoxide, albendazole sulfone, praziquantel and trans-4-hydroxypraziquantel in plasma was carried out by high-performance liquid chromatography-mass spectrometry ((LC-MS-MS). The plasma samples were prepared by liquid-liquid extraction using dichloromethane as extracting solvent. The partial HPLC resolution of drug and metabolites was obtained using a cyanopropyl column and a mobile phase consisting of methanol:water (3:7, v/v) plus 0.5% of acetic acid, at a flow rate of 1.0 mL/min. Multi reaction monitoring detection was performed by electrospray ionization in the positive ion mode, conferring additional selectivity to the method. Method validation showed relative standard deviation (precision) and relative errors (accuracy) lower than 15% for all analytes evaluated. The quantification limit was 5 ng/mL and the linear range was 5-2500 ng/mL for all analytes. The method was used for the determination of drug and metabolites in swine plasma samples and proved to be suitable for pharmacokinetic studies.     

A GC-MS method for the determination of isoxsuprine in biological fluids of the horse utilizing electron impact ionization

Isoxsuprine is used to treat navicular disease and other lower-limb problems in the horse. Isoxsuprine is regulated as a class 4 compound by the Association of Racing Commissioners, International (ARCI) and, thus, requires regulatory monitoring. A gas chromatography-mass spectrometry method utilizing electron impact ionization was developed and validated for the quantitation of isoxsuprine in equine plasma or equine urine. The method utilized robotic solid-phase extraction and tri-methyl silyl ether products of derivatization. Products were bis-trimethylsilyl (TMS) isoxsuprine and tris-TMS ritodrine, which released intense quantifier ions m/z 178 for isoxsuprine and m/z 236 for ritodrine that were products of C-C cleavage. To our knowledge, this procedure is faster and more sensitive than other methods in the literature. Concentrations in urine and plasma of isoxsuprine were determined from a calibrator curve that was generated along with unknowns. Ritodrine was used as an internal standard and was, therefore, present in all samples, standards, and blanks. Validation data was also collected. The limit of detection of isoxsuprine in plasma was determined to be 2 ng/mL, the limit of quantitation of isoxsuprine in plasma was determined to be < 5 ng/mL. The mean coefficient of determination for the calibrator curves for plasma was 0.9925 +/- 0.0052 and for calibrator curves for urine 0.9904 +/- 0.0075. The recovery efficiencies at concentrations of 50, 200, and 300 ng/mL were 76%, 73%, and 76%, respectively, in plasma and 92%, 89%, and 91% in urine.     

GC-MS confirmation of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone in urine.

A procedure for the simultaneous confirmation of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone in urine specimens by gas chromatography-mass spectrometry (GC-MS) is described. After the addition of nalorphine and naltrexone as the two internal standards, the urine is hydrolyzed overnight with beta-glucuronidase from E. coli. The urine is adjusted to pH 9 and extracted with 8% trifluoroethanol in methylene dichloride. After evaporating the organic, the residue is sequentially derivatized with 2% methoxyamine in pyridine, then with propionic anhydride. The ketone groups on hydrocodone, hydromorphone, oxycodone, oxymorphone, and naltrexone are converted to their respective methoximes. Available hydroxyl groups on the O3 and O6 positions are converted to propionic esters. After a brief purification step, the extracts are analyzed by GC-MS using full scan electron impact ionization. Nalorphine is used as the internal standard for codeine, morphine, and 6-acetylmorphine; naltrexone is used as the internal standard for the 6-keto-opioids. The method is linear to 2000 ng/mL for the 6-keto-opioids and to 5000 ng/mL for the others. The limit of quantitation is 25 ng/mL in hydrolyzed urine. Day-to-day precision at 300 and 1500 ng/mL ranged between 6 and 10.9%. The coefficients of variation for 6-acetylmorphine were 12% at both 30 and 150 ng/mL. A list of 38 other basic drugs or metabolites detected by this method is tabulated.     

A sensitive liquid chromatography and mass spectrometry method for the determination of posaconazole in human plasma

Posaconazole is a novel extended-spectrum triazole that has favorable in vitro, in vivo and clinical activity against a number of yeasts and moulds. Posaconazole is available as an oral suspension. The dosage found to result in monitored plasma levels that correlate with clinical evidence of good antifungal activity is 800 mg/day in divided doses. A liquid chromatographic/mass spectrometric method (LC-MS/MS) that can be used by clinicians wishing to quantitate, and thereby monitor, plasma levels of posaconazole in certain patients was validated. The method utilized semi-automated 96-well protein precipitation with gradient chromatographic separation of analytes using a Varian Polaris C-18A (2.0 mm x 50 mm, 5-microm particle size) column. The approximate retention time of posaconazole was 2.0 min. Analytes were detected by using tandem mass spectrometry. Sample introduction and ionization was performed by atmospheric pressure chemical ionization in the positive-ion mode. This method has been proven suitable for routine quantitation of posaconazole over the concentration range of 5.00-5000 ng/mL. Inter-run precision based on percent relative deviation for replicate quality controls was < or = 6.2%. Inter-run accuracy expressed as %DIFF was +/-4.0%. Posaconazole quality controls were stable in human plasma for up to five freeze-thaw cycles, when frozen at -20 degrees C for at least 105 days and when kept at room temperature for 24 h. The lower limit of quantitation was 5.00 ng/mL for a 100-microL sample aliquot. These data indicate that the LC-MS/MS method described is suitable for the rapid measurement of posaconazole over the concentration range of 5.00-5000 ng/mL.     

Determination of 3'-C-ethynylcytidine in human plasma and urine by liquid chromatographic-electrospray ionization tandem mass spectrometry

A liquid chromatographic-electrospray ionization tandem mass spectrometric (LC-ESI/MS/MS) method was developed for the quantitative analysis of a novel anticancer drug, 3'-C-ethynylcytidine (I) in human plasma and urine. I and its stable isotope-labeled internal standard (II) were extracted from human plasma and urine samples using a polymer-based cation-exchange cartridge, and LC-ESI/MS/MS analysis was performed by monitoring the positive fragment ions of I and II. The linear ranges are 1-500 ng/ml in plasma and 10-5000 ng/ml in urine. The limits of quantitation for I were 1 ng/ml in plasma and 10 ng/ml in urine. The relative errors (RE) for I ranged from -8.4 to 3.0% in plasma and from 0.8 to 4.4% in urine. The relative standard deviations (RSD) for I ranged from 1.2 to 8.9% in plasma and from 0.7 to 2.8% in urine. This validated analytical method is demonstrated to be useful for the analysis of I in human plasma and urine in clinical studies.     

采用正负离子切换模式的LC-ESI-MS/MS法测定大鼠灌胃给药后京尼平苷的血浆浓度(英文)

建立了一种快速、专属性强的液相色谱-串联质谱方法(LC-MS/MS)测定中药栀子的主要活性成分–京尼平苷的大鼠血药浓度去评价其临床前药动学特征。京尼平苷血浆样品经沉淀蛋白后,采用DiamonsilC18色谱柱进行分析,采用流动相10mM醋酸铵–甲醇(20:80,v/v),流速0.6mL/min。样品测定采用"Truncated"多反应检测模式,采用正离子m/z411→411测定京尼平苷,采用负离子m/z415→295测定内标葛根素。线性浓度范围为10.0–5000ng/mL,最低定量下限为10.0ng/mL,样品提取回收率范围为84.8%–90.5%。该确证方法成功应用于大鼠灌胃(给药剂量200mg/kg)给予京尼平苷后的药动学研究。     

Liquid chromatography-tandem mass spectrometry screening method for the simultaneous detection of stimulants and diuretics in urine

This study established a simultaneous screening method based on solid-phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS-MS) for the detection of 23 stimulants and 23 diuretics in human urine. An electrospray ionization source and multiple reaction monitoring were used for data acquisition. All stimulants and diuretics were separated in less than 12.52 min. The limits of detection were in the range of 25-500 ng/mL for stimulants and 25-125 ng/mL for diuretics. To evaluate the performance of this method, urine samples were collected from 1627 athletes in Taiwan, and 7 positive samples were found. This LC-MS-MS method not only meets the minimum required performance limits set by the World Anti-Doping Agency but also provides a fast way to analyze the authentic urine samples in doping control laboratories.     

Simultaneous analysis of the Delta9-THC metabolites 11-nor-9-carboxy-Delta9-THC and 11-hydroxy-Delta9-THC in meconium by GC-MS

Neonates that are exposed to cannabinoids in utero may have characteristic physical and mental developmental problems throughout their lives. The early identification of exposed neonates allows early intervention and anticipation of potential problems. Testing meconium detects maternal marijuana use over the last four months of gestation, providing a better drug exposure marker than urine. However, the distribution of metabolites in meconium is not identical to urine and analytical methods must be adapted. Both the major urine metabolite, 11-nor-9-carboxy-Delta9-tetrahydrocannabinol (9-carboxy-THC), and a minor urine metabolite, 11-hydroxy-Delta9-tetrahydrocannabinol (11-hydroxy-THC), are common in meconium. Currently published methods to extract these two metabolites for instrumental analysis are time-consuming and laborious, often involving the preparation of two fractions. This study describes a simple solid-phase extraction method and an optimized hydrolysis method that allow the preparation and analysis of both metabolites in a single extract. The limit of detection by this extraction method was 5 ng/g for both metabolites with an analytical measurement range from 10 to 500 ng/g. The recovery at 100 ng/g was greater than 62% for both analytes. The analysis of 246 cannabinoid screen positive specimens illustrated the importance of including the 11-hydroxy-THC in a meconium marijuana confirmation: 16 specimens confirmed positive for 11-hydroxy-THC only, resulting in a 6.5% increase in the positivity rate compared to 9-carboxy-THC alone.     

LC—MS—MS法测定人体血浆中左炔诺孕酮浓度的方法学研究

目的建立人体血浆中左炔诺孕酮浓度测定的LC-MS-MS法。方法血浆中目标成分采用乙酸乙酯萃取。色谱柱为BDSHypersil C18柱（3μm，2．1mm×50mm），以水（含0．5‰甲酸）-乙腈-甲醇（18：32：50）为流动相，流速为0．20mL·min^-1，采用ESI^＋MRM方式进行离子监测。质谱检测条件：离子源电压5．0kV，加热毛细管温度：300℃，鞘气（N2）流速：20L·Min^-1，辅助气流速：2L·min^-1。结果血浆中左炔诺孕酮在0．313～40．0ng·mL^-1。线性关系良好（r=0．9993），最低定量限为0．313ng·mL^-1，方法回收率为91．0％～105．7％，萃取回收率为77．2％～80．8％，日内及日间RSD均〈15％。结论本方法灵敏、准确，适用于临床试验中生物样品定量分析。     

Selective,sensitive, and rapid liquid chromatography-tandem mass spectrometry method for determination of Guaifenesin in human plasma

A simple, sensitive, selective, and rapid liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed and validated for quantitation of guaifenesin in human plasma using guaifenesin-d3 as internal standard (IS). Following solid phase extraction, the analyte was chromatographed using an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by MS in multiple reaction-monitoring (MRM) mode (positive ion mode). The limit of quantitation for this method was 8?ng/mL and the linear dynamic range was 8–2200?ng/mL.