A sensitive assay for the quantification of morphine and its active metabolites in human plasma and dried blood spots using high-performance liquid chromatography–tandem mass spectrometry

Opioids such as morphine are the cornerstone of pain treatment. The challenge of measuring the concentrations of morphine and its active metabolites in order to assess human pharmacokinetics and monitor therapeutic drugs in children requires assays with high sensitivity in small blood volumes. We developed and validated a semi-automated LC-MS/MS assay for the simultaneous quantification of morphine and its active metabolites morphine 3β-glucuronide (M3G) and morphine 6β-glucuronide (M6G) in human plasma and in dried blood spots (DBS). Reconstitution in water (DBS only) and addition of a protein precipitation solution containing the internal standards were the only manual steps. Morphine and its metabolites were separated on a Kinetex 2.6-μm PFP analytical column using an acetonitrile/0.1% formic acid gradient. The analytes were detected in the positive multiple reaction mode. In plasma, the assay had the following performance characteristics: range of reliable response of 0.25–1000 ng/mL (r ² > 0.99) for morphine, 1–1,000 ng/mL (r ² > 0.99) for M3G, and 2.5–1,000 ng/mL for M6G. In DBS, the assay had a range of reliable response of 1–1,000 ng/mL (r ² > 0.99) for morphine and M3G, and of 2.5–1,000 ng/mL for M6G. For inter-day accuracy and precision for morphine, M3G and M6G were within 15% of the nominal values in both plasma and DBS. There was no carryover, ion suppression, or matrix interferences. The assay fulfilled all predefined acceptance criteria, and its sensitivity using DBS samples was adequate for the measurement of pediatric pharmacokinetic samples using a small blood of only 20–50 μL.     

On-line desalting and determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in microdialysis and plasma samples using column switching and liquid chromatography/tandem mass spectrometry

A sensitive and reproducible method for the determination of morphine and the metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) was developed. The method was validated for perfusion fluid used in microdialysis as well as for sheep and human plasma. A C18 guard column was used to desalt the samples before analytical separation on a ZIC HILIC (hydrophilic interaction chromatography) column and detection with tandem mass spectrometry (MS/MS). The mobile phases were 0.05% trifluoroacetic acid (TFA) for desalting and acetonitrile/5 mM ammonium acetate (70:30) for separation. Microdialysis samples (5 microL) were directly injected onto the system. The lower limits of quantification (LLOQ) for morphine, M3G and M6G were 0.50, 0.22 and 0.55 ng/mL, respectively, and the method was linear from LLOQ to 200 ng/mL. For plasma, a volume of 100 microL was precipitated with acetonitrile containing internal standards (deuterated morphine and metabolites). The supernatant was evaporated and reconstituted in 0.05% TFA before the desalting process. The LLOQs for sheep plasma were 2.0 and 3.1 ng/mL and the ranges were 2.0-2000 and 3.1-3100 ng/mL for morphine and M3G, respectively. For human plasma, the LLOQs were 0.78, 1.49 and 0.53 ng/mL and the ranges were 0.78-500, 1.49-1000 and 0.53-500 ng/mL for morphine, M3G and M6G, respectively.     

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography for analysis of morphine and its four metabolites in human urine

A cation-selective exhaustive injection and sweeping micellar EKC (CSEI-Sweep-MEKC) was established to analyze morphine and its four metabolites, including codeine, normorphine (NM), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G). After SPE, the urine samples were analyzed by this CE method. The phosphate buffer (75 mM, pH 2.5) containing 30% methanol was first filled into an uncoated fused-silica capillary (40 cm, 50 microm id), then a high-conductivity buffer (120 mM phosphate, 10.3 kPa for 99.9 s) followed. The pretreated urine sample was loaded by electrokinetic injection (10 kV, 600 s). The stacking and separation were performed by using phosphate buffer (25 mM, pH 2.5) containing 22% methanol and 100 mM SDS at -20 kV, and detected at 200 nm. During method validation, calibration plots were linear (r > or = 0.998) over a range of 30-3000 ng/mL for morphine, NM, and codeine, 100-2000 ng/mL for M6G, and 80-3200 ng/mL for M3G. The LODs (S/N = 5, sampling 600 s at 10 kV) were 10 ng/mL for morphine, NM, and codeine, 35 ng/mL for M6G, and 25 ng/mL for M3G. This stacking CE method could increase 2500-fold sensitivity of codeine, when comparing with CZE. Five addicts' urine specimens were analyzed. Their results were compared with those of LC-MS-MS, and showed good coincidence. This method could be feasible for monitoring morphine and its metabolites in forensic interest and pharmacokinetic investigations.     

Portable Immuno-Microchip Analyzer for the Determination of Morphine and Its Analogs

A portable immuno-microchip system consisting of low-cost biocompatible-polystyrene chips, a mini-vacuum pump, and a base unit for a LED light source as a miniaturized optical readout was devised. The surfaces of the micro-channels were processed with plasma to be more hydrophilic and were characterized by kinetic coating with a CCD scanning method. To demonstrate the feasibility of this novel system for the determination of a small molecule control assay, a simple, specific, and rapid method was established for drug analysis of morphine and its analogs, 6-monoacetylmorphine and morphine-3-glucuronide. Morphine-3-site antigen and a multi-target polyclonal antibody to morphine and derivatives were prepared and a competitive immunoassay was performed on the chip. The measurement can be carried out automatically and avoids time-consuming incubation steps. A detection sensitivity of below 1.0 ng/mL was achieved, which is comparable with the enzyme-linked immunosorbent assay (ELISA) technique.     

Method for quantification of morphine and its 3- and 6- glucuronides, codeine, codeine glucuronide and 6-monoacetylmorphine in human blood by liquid chromatography-electrospray mass spectrometry for routine analysis in forensic toxicology.

Simultaneous determination of opiates and their glucuronides in body fluids has a great practical interest in the forensic assessment of heroin intoxication. A selective and sensitive method for quantification of morphine and its 3- and 6-glucuronides, codeine, codeine glucuronide and 6-monoacetylmorphine (6-MAM) based on liquid chromatography-electrospray ionisation mass spectrometry is described. The drugs were analysed in human autopsy whole blood after solid-phase extraction on a C8 cartridge. The separation was performed on an ODS column in acetonitrile (analysis time 15 min). For the quantitative analysis, deuterated analogues of each compound were used as internal standards. Selected-ion monitoring was applied where the molecular ion was chosen for quantification. The limits of quantification were 0.5 ng/ml for morphine and 6-MAM and 1 ng/ml for the 6-glucuronide of morphine, codeine-6-glucuronide and codeine and 5 ng/ml for the 3-glucuronide of morphine.     

Quantification of morphine and its major metabolites M3G and M6G in antemortem and postmortem samples

Morphine is one of the most effective agents for the control of significant pain, primarily metabolized to morphine‐3‐glucuronide (M3G) and morphine‐6‐glucuronide (M6G). While M6G is a potent opioid agonist, M3G has no opioid action and seems to have a role in side‐effects caused by morphine. In this study, a reversed‐phase high‐performance liquid chromatographic method with diode‐array and electrochemical detection was developed for the simultaneous determination of morphine, M3G and M6G in antemortem and postmortem samples (plasma, whole blood, urine, liver, kidney and brain). Morphine, glucuronides and internal standard were extracted by double solid‐phase extraction and the separation was carried out with a Waters Spherisorb® ODS2 reversed‐phase column and potassium phosphate buffer (pH = 2.2)–acetonitrile containing sodium dodecyl sulfate as the mobile phase. The method proved to be specific with good linearity for all analytes in a calibration range from 1 to 600 ng/mL and proved to be accurate and have adequate precision and recovery. Limits of detection in the studied matrices were 0.4–4.5 ng/mL for morphine, 2.7–6.1 ng/mL for M3G and 0.8–4.4 ng/mL for M6G. The proposed method can be successfully applied to quantify morphine and its metabolites in several biological samples, covering the major routes of distribution, metabolism and elimination of morphine. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantitative gas chromatographic/mass spectrometric analysis of morphine glucuronides in human plasma by negative ion chemical ionization mass spectrometry

A sensitive and specific method for the determination of morphine glucuronides in human plasma is presented. Morphine glucuronides, namely morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G), were extracted from plasma by solid-phase extraction on C(18) cartridges at pH 9.3 and derivatized to their pentafluorobenzyl ester trimethylsilyl ether derivatives. The compounds were measured by gas chromatography/negative ion chemical ionization mass spectrometry without any further purification. Using this detection mode, a diagnostic useful fragment ion at m/z 748 was obtained at high relative abundance for both target compounds. [(2)H(3)]-labeled morphine glucuronides were used as internal standards. Calibration graphs were calculated by polynomial fit within a range of 10-1280 and 15-1920 nmol l(-1) for the 6- and 3-glucuronide, respectively. At the limit of quantitation (LOQ), the inter-assay precision was 2.21% (M3G) and 2.23% (M6G) and the GC/MS assay variability was 1.8% (M3G) and 0.9% (M6G). The accuracy at the LOQ showed deviations of +4.92% (M3G) and +1.5% (M6G). The sample recovery after solid-phase extraction was 84.7% for both M3G and M6G. The method is rugged, rapid and robust and has been applied to the batch analysis of morphine glucuronides during pharmacokinetic profiling of the drugs.     

Characterization and application of quantum dot nanocrystal–monoclonal antibody conjugates for the determination of sulfamethazine in milk by fluoroimmunoassay

Quantum dot (Qdot) nanocrystals have been increasingly used as fluorescence labels in fluoroimmunoassays recently because of their excellent optical characteristics. In this paper, a new monoclonal antibody (MAb) against sulfamethazine (SMZ) was successfully produced and linked to Qdot nanocrystals by covalent coupling. The Qdot–MAb conjugates were characterized by SDS-PAGE and high-performance capillary electrophoresis (HPCE). An enzyme-linked immunosorbent assay (ELISA) method was utilized to evaluate the antigen–antibody binding affinity and then a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) for the detection of SMZ in milk by using Qdots as fluorescent labels was evaluated. The results showed that the 50% inhibition values (IC₅₀) of the cFLISA were 4.3 ng/mL in milk and 5.2 ng/mL in PBS, and the limits of detection (LODs) were 0.6 ng/mL in milk and 0.4 ng/mL in PBS, respectively. The recoveries of SMZ from spiked milk samples at levels of 10–100 ng/mL ranged from 94 to 106%, with coefficients of variation (CVs) of 2.1–9.2%. Figure Shematic diagram of the direct cFLISA procedure Jianzhong Shen and Fei Xu contributed equally to this work.     

Ultrafast liquid chromatography/tandem mass spectrometry bioanalysis of polar analytes using packed silica columns

Ultrafast liquid chromatography/tandem mass spectrometry (LC/MS/MS) bioanalysis was demonstrated with the use of packed silica columns operated under elevated flow rates. A special effort has been made to achieve ultrafast analysis without sacrificing chromatographic resolution. Two multiple analyte/metabolites assays, (1) morphine/morphine-6-glucuronide(M6G)/morphine-3-glucuronide(M3G) and (2) midazolam/1'-hydroxymidazolam/4-hydroxymidazolam, were used to demonstrate the speed, sensitivity, peak shape and separation of the ultrafast methods utilizing silica columns. In both methods adequate chromatographic separation was a necessity because quantitation results would be otherwise compromised due to cross interference between different selected reaction monitoring (SRM) transitions. Baseline resolutions between morphine, M6G and M3G in human plasma extracts were achieved within 30 s on a 50 x 3 mm Betasil silica column operated at 4 mL/min of isocratic acetonitrile/water mobile phase. The total injection-to-injection cycle time was 48 s with a simple, single-autosampler/single-column setup, when a Shimadzu SIL-HT autosampler was used. Baseline resolution between 1'-hydroxymidazolam and 4-hydroxymidalolam in monkey plasma extracts was achieved within 33 s using similar conditions. Due to the absence of carry-over in this case, no rinsing of the injection needle was necessary, resulting in a cycle time of only 39 s/sample. These ultrafast methods were successfully used to analyze extracted biological samples and proved to be reproducible, reliable and generated equivalent pharmaco-kinetic (PK) results to those obtained by regular flow LC/MS/MS analysis to support discovery PK studies.     

Fast reliable assay for morphine and its metabolites using high-performance liquid chromatography and native fluorescence detection

A method for the fast analysis of morphine (M), normorphine (NM), morphine-3- and -6-glucuronides (M3G and M6G) and codeine (C) is described which has the advantages of sensitivity, speed and specificity. Dihydrocodeine and heroin can also be assayed. The method is based on extraction of the opiates from serum, plasma and cerebrospinal fluid using reversed-phase solid-phase extraction columns, followed by reversed-phase high-performance liquid chromatography with native fluorescence detection. The extraction step provides greater than 95% recovery, and the response of the detection system is linear from 0.5 to beyond 750 ng. The method allows analysis of M, NM, M3G, M6G and C. No other drugs have been found to interfere with the assay. The assay offers a quick, cheap and reliable method of specifically determining morphine and its metabolites, including the potent M6G, from a small sample volume; this will be of advantage to both clinician and basic scientist.     

Simultaneous analysis of gemfibrozil,morphine, and its two active metabolites in different mouse brain structures using solid‐phase extraction with ultra‐high performance liquid chromatography and tandem mass spectrometry with a deuterated internal standard

A rapid and sensitive bioassay was established and validated to simultaneously determine gemfibrozil, morphine, morphine‐3β‐glucuronide, and morphine‐6β‐glucuronide in mouse cerebrum, epencephalon, and hippocampus based on ultra‐high performance liquid chromatography and tandem mass spectrometry. The deuterated internal standard, M6G‐d3, was mixed with the prepared samples at 10 ng/mL as the final concentration. The samples were transferred into the C₁₈ solid‐phase extraction columns with gradient elution for solid‐phase extraction. The mobile phase consisted of methanol and 0.05% formic acid (pH 3.2). Multiple reaction monitoring has been applied to analyze gemfibrozil (m/z 249.0 → 121.0) in anion mode, and M6G‐d3 (m/z 465.1 → 289.1), morphine (m/z 286.0 → 200.9), and M3G and M6G (m/z 462.1 → 286.1) in the positive ion mode. The method has a linear calibration range from 0.05 to 10 ng for gemfibrozil, morphine, and M3G and M6G with correlation coefficients >0.993. The lower limit of quantitation for all four analytes was 0.05 ng/mL, relative standard deviation of intra‐ and interday precision was less than 10.5%, and the relative error of accuracy was from ?8.2 to 8.3% at low, medium, and high concentrations for all the analytes. In conclusion, gemfibrozil can influence the morphine antinociception after coronary heart disease induced chronic angina by the change in one of morphine metabolites', M3G, distribution in mouse brain.     

海洛因滥用者唾液中毒品代谢物的SPE-GC/MS-SIM分析

本文首先考查了不同类型不同厂家的固相萃取柱的萃取效果,采用提取效果较好的50 mg/mL的Clean-screen CSDAUL01混合型强阳离子固相萃取柱,对唾液中的吗啡、可待因、6-单乙酰吗啡等3种鸦片类毒品进行提取后,将提取液吹干;再经MBTFA衍生化后,进行GC/MS-SIM检测。以乙基吗啡为内标,3种毒品的线性相关系数均大于0.99,线性范围为10～1000 ng/mL,相对回收率分别为85%～110%、94%～107%和75%～92%;相对标准偏差小于10%。3种毒品的检测限分别为2 ng/mL、1 ng/mL和2 ng/mL。该方法灵敏度高、重现性好、操作简便,可用于鸦片类毒品滥用者及中毒者唾液中的毒品及其代谢物的检测。     

Analysis of codeine, dihydrocodeine and their glucuronides in human urine by electrokinetic capillary immunoassays and capillary electrophoresis-ion trap mass spectrometry

Screening for and confirmation of illicit, abused and banned drugs in human urine is a timely topic in which capillary separation techniques play a key role. Capillary electrophoresis (CE) represents the newest technology employed in this field of analysis. Two rapid competitive binding, electrokinetic capillary-based immunoassays are shown to be capable of recognizing the presence, but not the identity, of urinary opioids, namely codeine (COD), codeine-6-glucuronide, dihydrocodeine (DHC), dihydrocodeine-6-glucuronide, morphine (MOR), morphine-3-glucuronide and ethylmorphine (EMOR). In these approaches, aliquots of urine and immunoreagents of a commercial, broadly cross-reacting fluorescence polarization immunoassay for opiates were combined and analyzed by capillary zone electrophoresis or micellar electrokinetic capillary chromatography with laser induced fluorescence detection. With the fluorescent tracer solution employed, the former method is shown to provide simple electropherograms which are characterized by an opioid concentration dependent magnitude of the free tracer peak. In presence of dodecyl sulfate micelles, however, two tracer peaks with equal opioid concentration sensitivity are monitored. These data suggest the presence of two fluorescent tracers which react competitively with the urinary opioids for the binding sites of the antibody. Assay sensitivities for COD and MOR are comparable (10 ng/ml), whereas those for DHC and EMOR are about four-fold lower. Furthermore, glucuronides are shown to react like the corresponding free opioids. Analysis of urines that were collected after administration of 7 mg COD and 25 mg DHC tested positively in both assay formats. The presence of the free and conjugated codeinoids in these urines and their identification was accomplished by capillary electrophoresis-ion trap mass spectrometry (CE-MS). This confirmatory assay is based upon solid-phase extraction using a mixed-mode polymer cartridge followed by CE hyphenated to the LCQ mass spectrometer with electrospray ionization in the positive ion mode. With this technology, MS2 is employed for proper identification of COD (m/z 300.4) and DHC (m/z 302.4) whereas MS3 provides unambiguous identification of the glucuronides of COD (m/z 476.5) and DHC (m/z 478.5) via their fragmentation to COD and DHC, respectively. MSn (n > or = 2) is shown to be capable of properly identifying the urinary codeinoids on the 100-200 ng/ml concentration level.     

Development of Monoclonal Antibody-based Enzyme-linked Immunosorbent Assay to the Estrogen Diethylstilbestrol

Diethylstilbestrol （DES） is a synthetic estrogen that has ever been used worldwide. Polyclonal antibodies （PAbs） were used in immunoassay for detection of DES residues in environmental and agricultural samples in previous paper. In this paper, an indirect competitive enzyme-linked immunosorbent assay （icELISA） was developed based on monoclonal antibody （MAb） for the determination of diethylstilbestrol. Mono-o-carboxypropyldiethylstilbestrol （DES-CP） and mono-o-carboxymethyldiethylstilbestrol （DES-CME） were synthesized to be haptens. DES-CP was coupled to bovine serum albumin （BSA） to be an immunogen in BALB/c female mouse for MAb production. The MAb was characterized for specificity and affinity to DES in icELISA. Under the optimum condition, the icELISA showed an ICs0 of 9.8 ng/mL, the limit of detection （IC20） of 2.3 ng/mL and a working range of 2-42 ng/mL. Hexestrol and dienestrol exhibited cross-reactivity values were 44% and 27%, respectively. Cross-reactivity of natural estrogen 17β-estradiol was less than 0.1%. The influences of some factors such as salt concentration, pH and organic solvent concentration on the assay were evaluated. The concentrations of DES in the fortified water samples determined by the assay were correlated well with the fortification levels. The results were conf＇m＇ned with analysis by HPLC.     

High-performance liquid chromatographic determination of morphine and its 3- and 6-glucuronide metabolites: improvements to the method and application to stability studies

Improvements to previously reported methods for the determination of morphine, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in human plasma are described. The improved methods involve the use of a solid-phase extraction cartridge and a chromatographic system which uses paired-ion reversed-phase high-performance liquid chromatography with a radially compressed column. Only one cartridge is used to prepare each sample for chromatography and each cartridge may be used for at least fourteen 1-ml plasma samples. The recovery is greater than 85%. The improvements to the method of sample pretreatment and in the chromatographic conditions have allowed determination of morphine, M3G and M6G in human plasma down to 13.3 nmol/l (coefficient of variation = 9.3%), 108 nmol/l (6.6%) and 41 nmol/l (6.7%), respectively, using ultraviolet detection alone. It was shown that all three compounds were stable in plasma for up to 101 weeks when stored at -20 degrees C.     

Development of an Ultra-sensitive Chemiluminescence Enzyme Immunoassay for the Determination of Diethylstilbestrol in Seafood

An ultra-sensitive indirect competitive chemiluminescence enzyme immunoassay was developed for screening diethylstilbestrol in fish and shrimp samples. The concentration of diethylstilbestrol that caused 50% inhibition of the binding enzyme marker (IC₅₀) was 0.32 ng/mL and the limit of detection was 0.0068 ng/mL; the linear range was from 0.028 ng/mL to 3.60 ng/mL. The assay showed cross-reactivity of 7.1% and 2.8% with dienestrol and hexoestrol, respectively, but negligible cross-reactivity with estradiol, estrone, ethinyloestradiol, and progestin. The recovery from spiked fish and shrimp samples varied from 68.5% to 92.5%, and the mean coefficients of variation within groups and between groups were 6.2% and 8.0%, respectively. Our results indicated that the assay is a simple, sensitive, specific, and accurate method for screening fish and shrimp samples for diethylstilbestrol.     

Simultaneous determination of morphine‐6‐d‐glucuronide,morphine‐3‐d‐glucuronide and morphine in human plasma and urine by ultra‐performance liquid chromatography–tandem mass spectrometry: Application to M6G injection pharmacokinetic study

A robust ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the determination of morphine‐6‐d ‐glucuronide (M6G), morphine‐3‐d ‐glucuronide (M3G) and morphine (MOR) in human plasma and urine has been developed and validated. The analytes of interest were extracted from plasma by protein precipitation. The urine sample was prepared by dilution. Both plasma and urine samples were chromatographed on an Acquity UPLC HSS T₃ column using gradient elution. Detection was performed on a Xevo TQ‐S tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. Matrix interferences were not observed at the retention time of the analytes and internal standard, naloxone‐D5. The lower limits of quantitation of plasma and urine were 2/0.5/0.5 and 20/4/2 ng/mL for M6G/M3G/MOR, respectively. Calibration curves were linear over the concentration ranges of 2–2000/0.5–500/0.5–500 and 20–20,000/4–4000/2–2000 ng/mL for M6G/M3G/MOR in plasma and urine samples, respectively. The precision was <7.14% and the accuracy was within 85–115%. Furthermore, stability of the analytes at various conditions, dilution integrity, extraction recovery and matrix effect were assessed. Finally, this quantitative method was successfully applied to the pharmacokinetic study of M6G injection in Chinese noncancer pain patients.     

Validation of a HPLC/MS method for simultaneous quantification of clonidine,morphine and its metabolites in human plasma

A high‐performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantification of morphine, morphine's major metabolites morphine‐3‐glucuronide and morphine‐6‐glucuronide, and clonidine, to support the pharmacokinetic analysis of an ongoing double‐blinded randomized clinical trial that compares the use of morphine and clonidine in infants diagnosed with neonatal abstinence syndrome. Plasma samples were processed by solid‐phase extraction and separated on an Inertsil ODS‐3 (4 μm) column using an 0.1% formic acid in water–0.1% formic acid in methanol gradient. Detection of the analytes was conducted in the positive multiple reaction monitoring mode. The range of quantitation was 1–1000 ng/mL for morphine, morphine‐3‐glucuronide and morphine‐6‐glucuronide, and 0.25–100 ng/mL for clonidine. Intra‐day and inter‐day accuracy and precision were ≤15% for all analytes across the quantitation range. Extraction recovery rates were ≥94% for morphine, ≥90% for M3G, ≥87% for M6G and ≥ 79% for clonidine. Matrix effect ranged from 85–94% for clonidine to 101–106% for M3G. The method fulfilled all predetermined acceptance criteria and required only 100 μL of starting plasma volume. Furthermore, it was successfully applied to 30 clinical trial plasma samples.     

Direct determination of estriol 3- and 16-glucuronides in pregnancy urine by column-switching liquid chromatography with electrospray tandem mass spectrometry

Using column-switching liquid chromatography/tandem mass spectrometry (LC-MS/MS), we developed an improved analytical method of urinary estriol glucuronides. This new method is derived predominantly from maternal and fetal precursors in pregnancy. We used in the following procedure: first, we filtered urine samples with a membrane filter. Next, we directly injected the 50 microL aliquot of urine samples onto a pre-column. Then, after activating the column-switching valve, we backflushed the loaded samples onto the C(18) analytical column. Urine samples can be assayed within 20 min without any sample preparation steps. We monitored separated estriol glucuronides by negative electrospray ionization (ESI) and selected-reaction monitoring (SRM). The calibration range of estriol-3-glucuronide (E3-3G) and estriol-16-glucuronide (E3-16G) was 0.1-20 microg/mL and the linearity of the method was 0.9984 for E3-3G and 0.9987 for E3-16G. The limits of detection at a signal-to-noise (S/N) ratio of 3 were 10 ng/mL (E3-3G) and 5 ng/mL (E3-16G). The analytical recovery was over 85% and, in general, inter-day and intra-day variability for precision and accuracy were less than 10%. When applied to a pregnancy urine sample to biomedical monitoring of the function of the maternal/fetal unit, the proposed method allowed rapid and sensitive screening for the detection of E3-3G and E3-16G.     

Use of gas chromatography/mass spectrometry with positive chemical ionization for the determination of opiates in human oral fluid

An analytical method for the simultaneous determination of codeine, morphine and 6-acetylmorphine (6AM) in human oral fluid was developed. The method involves liquid-liquid extraction in Toxitubes A, derivatization with 99:1 (v/v) N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS), and gas chromatography/mass spectrometry with positive chemical ionization (GC/PCI-MS) determination. The detector response was linear over the concentration range 30-500 ng/mL with coefficients of correlation higher than 0.99. The precision was acceptable with coefficients of variation less than 7.5%. The limits of detection achieved were 0.7 ng/mL for codeine, 2.0 ng/mL for morphine, and 0.6 ng/mL for 6AM. The method proposed was applied to 80 oral fluid samples from opiates users, 98% of which were positive for the three analytes. Human oral fluid is a suitable biological fluid for the determination of opiates by GC/PCI-MS.