Quantification of dialkylphosphate metabolites of organophosphorus insecticides in human urine using 96-well plate sample preparation and high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry

Organophosphorus (OP) pesticides kill by disrupting a targeted pest's brain and nervous systems. But if humans and other animals are sufficiently exposed, OP pesticides can have the same effect on them. We developed a fast and accurate high-performance liquid chromatography–tandem mass spectrometry method for the quantitative measurement of the following six common dialkylphosphate (DAP) metabolites of organophosphorus insecticides: dimethylphosphate (DMP), dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate, (DEP), diethylthiophosphate (DETP), and diethyldithiophosphate (DEDTP). The general sample preparation included 96-well plate solid phase extraction using weak anion exchange cartridges. The analytical separation was performed by high-performance liquid chromatography with a HILIC column. Detection involved a triple quadrupole mass spectrometer with an ESI probe in negative ion mode using multiple reaction monitoring. Repeated analyses of urine samples spiked at 150, 90 and 32 ng/mL with the analytes gave relative standard deviations of less than 22%. The extraction efficiency ranged from 40% to 98%. The limits of detection were in the range of 0.04–1.5 ng/mL. The throughput is 1152 samples per week, effectively quadrupling our previous throughput. The method is safe, quick, and sensitive enough to be used in environmental and emergency biological monitoring of occupational and nonoccupational exposure to organophosphates.     

Revised method for routine determination of urinary dialkyl phosphates using gas chromatography–mass spectrometry

Among urinary organophosphorus pesticide (OP) metabolites, dialkyl phosphates (DAPs) have been most often measured as a sensitive biomarker in non-occupational and occupational OP exposure risk assessment. In our conventional method, we have employed a procedure including simple liquid–liquid extraction (diethyl ether/acetonitrile), derivatization (pentafluorobenzylbromide, PFBBr) and clean-up (multi-layer column) for gas chromatography–mass spectrometry (GC–MS) analysis starting from 5-mL urine samples. In this study, we introduce a revised analytical method for urinary DAPs; its main modification was aimed at improving the pre-derivatization dehydration procedure. The limits of detection were approximately 0.15 μg/L for dimethylphosphate (DMP), 0.07 μg/L for diethylphosphate (DEP), and 0.05 μg/L for both dimethylthiophosphate (DMTP) and diethylthiophosphate (DETP) in 2.5-mL human urine samples. Within-run precision (percent of relative standard deviation, %RSD) at the DAP levels varying in the range of 0.5–50 μg/L was 6.0–19.1% for DMP, 3.6–18.3% for DEP, 8.0–25.6% for DMTP and 9.6–27.8% for DETP. Between-run precision at 5 μg/L was below 15.7% for all DAPs. The revised method proved to be feasible to routine biological monitoring not only for occupational OP exposure but also for environmental background levels in the general population. Compared to our previous method, the revised method underscores the importance of adding pre-derivatization anhydration for higher sensitivity and precision.     

Determination of dialkyl phosphates in human hair for the biomonitoring of exposure to organophosphate pesticides

A new, simple, fast and sensitive method that enables the measurement of four dialkyl phosphates (DAPs) in human head hair is presented in the current study. The dialkyl phosphates, dimethyl phosphate (DMP), diethyl phosphate (DEP), diethyl thiophosphate (DETP) and diethyl dithiophosphate (DEDTP) are non-selective metabolites of the organophosphate pesticides (OPs). The extraction of DAPs from hair matrix was achieved by one step methanolic extraction. Head hair samples from general population and population occupationally exposed to OPs were analysed using gas chromatography–mass spectrometry (GC–MS) after derivatization with pentafluorobenzylbromide. The recovery of the target compounds was estimated at 84.3% for DMP, 116.1% for DEP, 109.0% for DETP and 91.5% for DEDTP. The limit of quantitation (LOQ) and detection (LOD) was 20 and 6 pg/mg for DMP, 10 and 5 pg/mg for DEP and DETP and 5 and 3 pg/mg for DEDTP, respectively. With-run and between-run precision as well as accuracy was estimated. The percentage of positive hair samples for DMP, DEP, DETP and DEDTP for the group of general population was 63.0%, 96.3%, 66.7%, and 70.4% respectively. The samples from the group with occupational exposure were positive for all dialkyl phosphates analysed. The median concentrations for DMP were 165.0 and 181.7 pg/mg, for DEP were 51.2 and 812.9 pg/mg, for DETP were 54.0 and 660.1 pg/mg, and for DEDTP were 40.0 and 60.6 pg/mg for the general population group and the group with occupational exposure respectively. Significant differences in the levels of the total dialkyl phosphates amongst exposed and not exposed groups were observed (p < 0.001). More specifically, the total ethyl phosphate (DEPs) and DAPs median concentrations were 119.5 and 301.5 pg/mg for the general population group and 1498.8 and 1694.4 pg/mg for the group with occupational exposure.     

Simultaneous determination of urinary dialkylphosphate metabolites of organophosphorus pesticides using gas chromatography-mass spectrometry

In this study, we developed a safe and sensitive method for the simultaneous determination of urinary dialkylphosphates (DAPs), metabolites of organophosphorus insecticides (OPs), including dimethylphosphate (DMP), diethylphosphate (DEP), dimethylthiophosphate (DMTP), and diethylthiophosphate (DETP), using a pentafluorobenzylbromide (PFBBr) derivatization and gas chromatography-mass spectrometry (GC-MS). Several parameters were investigated: pH on evaporation, reaction temperature and time for the derivatization, the use of an antioxidant for preventing oxidation, and a clean-up step. The pH was set at 6, adjusted with K2CO3, and the reaction temperature and time of derivatization were 80 degrees C and 30 min, respectively. Sodium disulfite was chosen as an antioxidant. The clean-up step was performed with a Florisil/PSE mini-column to remove the unreacted PFBBr and sample matrix. This established procedure markedly shortened the sample preparation time to only about 3 h, and completely inhibited the unwanted oxidization of dialkylthiophosphates. The limits of determination (LOD) were approximately 0.3 microg/L for DMP, and 0.1 microg/L for DEP, DMTP, and DETP in 5 mL of human urine. Within-series and between-day imprecision for the present method using pooled urine spiked with DAPs was less than 20.6% in the calibration range of 1-300 microg/L, and the mean recovery was 56.7-60.5% for DMP, 78.5-82.7% for DEP, 88.3-103.9% for DMTP, and 84.2-92.4% for DETP. This method detected geometric mean values of the urinary DAPs in Japanese with and without occupational exposure to OPs, 16.6 or 27.4 for DMP, 1.0 or 0.7 for DEP, 1.3 or 2.3 for DMTP, and 1.0 or 1.1 microg/L for DETP, respectively. The present method, which does not require special equipment except for GC-MS, is quick, safe, and sensitive enough to be adopted in routine biological monitoring of non-occupational as well as occupational exposure to OPs.     

Simultaneous determination of six dialkylphosphates in urine by liquid chromatography tandem mass spectrometry

Dialkylphosphates (DAP) are urinary markers of the exposure to organophosphates pesticides. The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitative determination of the following DAP: dimethylphosphate (DMP), dimethythiophosphate (DMTP), dimethyldithiophosphate (DMDTP), diethylphosphate (DEP), diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP). Dibutylphosphate (DBP) was used as internal standard. This method was based on a liquid-liquid extraction procedure, a chromatographic separation using an Inertsil ODS3 C18 column and mass spectrometric detection in the negative ion, multiple reaction monitoring (MRM) mode, following two ion transitions per compound. It yielded a limit of quantification of 2 microg/L for the six compounds and intra-assay coefficients of variation (CV%) lower than 20%. This method was applied to the analysis of urines samples from a small cohort of non-exposed volunteers. At least one of the six DAP was detected in each sample. This result confirmed the feasibility of a LC-MS/MS procedure for monitoring the general population exposure to some frequently employed organophosphate pesticides.     

Biological monitoring of occupational exposure to 5-fluorouracil: Urinary α-fluoro-β-alanine assay by high performance liquid chromatography tandem mass spectrometry in health care personnel

A new sensitive and specific HPLC–MS/MS method for the determination of α-fluoro-β-alanine (FBAL), the main metabolite of the antineoplastic drug 5-fluorouracil (5-FU), in urine for the biological monitoring survey of health care workers exposed to 5-FU is described. This procedure is characterized by a pre-column FBAL derivatization by 2,4-dinitrofluorobenzene followed by solid phase extraction sample clean-up. The chromatographic separation was achieved by hydrophilic interaction chromatography (HILIC) on a ZIC HILIC column (Sequant) and the quantification was performed by tandem mass spectrometry. The method offers high sensitivity with a quantification limit of 1 μg/l, which is an improvement on those previously reported. The within- and between-day precisions were less than 13% and 15% respectively at the LOQ and no significant relative matrix effect was observed for FBAL. The validated method was applied to the biological monitoring of occupational exposure to 5-FU in a French hospital. Pre- and post-shift urine samples were collected from 19 workers in a hospital pharmacy and an oncology ward over a period of 5 days. On a total of 121 analysed samples, measurable amounts of FBAL were detected in up to 29%, the concentrations range from LOQ to 22.7 μg/l, yielding evidence of occupational exposure to 5-FU. Such data are scarce and represent a step forward in assessing the occupational health risks associated with handling antineoplastic drugs.     

Diethylthiophosphate and diethyldithiophosphate induce genotoxicity in hepatic cell lines when activated by further biotransformation via Cytochrome P450

Organophosphorous (OP) compounds are the most commonly used pesticides. There are several published reports on the direct toxicity of OP pesticides, but few data on the toxicity of their metabolites. To determine if diethylthiophosphate (DETP) and diethyldithiophosphate (DEDTP), two of the major OP metabolites, demonstrate genotoxicity, and to elucidate the possible genotoxic mechanisms, we treated WRL68, HepG2, HeLa and human blood cells with different concentrations of DETP and DEDTP. We evaluated the possible contribution of oxidative stress generation and P450 enzymes to the genotoxicity of the OP metabolites, as determined using the comet assay. Our results showed that both OP metabolites (DETP and DEDTP) induce DNA damage only in the hepatic cell lines, and this effect could be related to a secondary non-diffusible metabolite generated by the activity of P450 enzymes since P450 enzyme inhibitors also inhibited the induction of DNA damage in hepatic cells. These secondary metabolites should be taken into account when assessing risk to human populations exposed to OP pesticides.     

Cytogenetic response without changes in peripheral cholinesterase enzymes following exposure to a sheep dip containing diazinon in vivo and in vitro

Occupational exposure to organophosphorus insecticides (OPs), such as diazinon, may be monitored by the measurement of the activity of peripheral cholinesterase enzymes, including erythrocyte acetylcholinesterase (EAChE) and plasma or serum cholinesterase (plasma or serum ChE). Exposures have also been measured by the analysis of dialkyl phosphate metabolites of OPs in urine. The potential health risks associated with exposure, especially those of a neurological nature, may then be estimated, and appropriate measures to reduce or eliminate exposures can be implemented. There is evidence that some OP pesticides may have in vivo genotoxic effects, suggesting a possible link with cancer with long term or repeated heavy exposures. This paper describes work performed in 17 subjects with a single or two exposures to a sheep dip containing diazinon. Urine samples revealed OP metabolites dimethylphosphate (DMP), dimethylthiophosphate (DMTP), diethylphosphate (DEP) and diethylthiophosphate (DETP) in 37% of subjects at low levels which were not elevated after exposure. EAChE and plasma ChE were also unchanged before and after exposure, and were similar to those measured in unexposed control groups. Sister chromatid exchanges (SCE), a marker of chromosome damage, was significantly elevated in peripheral blood lymphocytes after exposure compared with before. SCE were unchanged in a group of non-occupationally exposed workers. In vitro studies with both authentic diazinon (98%) and diazinon in a sheep dip formulation (45%) showed increased SCE and decreased replicative indices, suggesting toxic and genotoxic effects of diazinon.     

Cytogenetic response without changes in peripheral cholinesterase enzymes following exposure to a sheep dip containing diazinon in vivo and in vitro

Occupational exposure to organophosphorus insecticides (OPs), such as diazinon, may be monitored by the measurement of the activity of peripheral cholinesterase enzymes, including erythrocyte acetylcholinesterase (EAChE) and plasma or serum cholinesterase (plasma or serum ChE). Exposures have also been measured by the analysis of dialkyl phosphate metabolites of OPs in urine. The potential health risks associated with exposure, especially those of a neurological nature, may then be estimated, and appropriate measures to reduce or eliminate exposures can be implemented. There is evidence that some OP pesticides may have in vivo genotoxic effects, suggesting a possible link with cancer with long term or repeated heavy exposures. This paper describes work performed in 17 subjects with a single or two exposures to a sheep dip containing diazinon. Urine samples revealed OP metabolites dimethylphosphate (DMP), dimethylthiophosphate (DMTP), diethylphosphate (DEP) and diethylthiophosphate (DETP) in 37% of subjects at low levels which were not elevated after exposure. EAChE and plasma ChE were also unchanged before and after exposure, and were similar to those measured in unexposed control groups. Sister chromatid exchanges (SCE), a marker of chromosome damage, was significantly elevated in peripheral blood lymphocytes after exposure compared with before. SCE were unchanged in a group of non-occupationally exposed workers. In vitro studies with both authentic diazinon (98%) and diazinon in a sheep dip formulation (45%) showed increased SCE and decreased replicative indices, suggesting toxic and genotoxic effects of diazinon.     

Occupational exposure to HDI: Progress and challenges in biomarker analysis

1,6-Hexamethylene diisocyanate (HDI) is extensively used in the automotive repair industry and is a commonly reported cause of occupational asthma in industrialized populations. However, the exact pathological mechanism remains uncertain. Characterization and quantification of biomarkers resulting from HDI exposure can fill important knowledge gaps between exposure, susceptibility, and the rise of immunological reactions and sensitization leading to asthma. Here, we discuss existing challenges in HDI biomarker analysis including the quantification of N-acetyl-1,6-hexamethylene diamine (monoacetyl-HDA) and N,N′-diacetyl-1,6-hexamethylene diamine (diacetyl-HDA) in urine samples based on previously established methods for HDA analysis. In addition, we describe the optimization of reaction conditions for the synthesis of monoacetyl-HDA and diacetyl-HDA, and utilize these standards for the quantification of these metabolites in the urine of three occupationally exposed workers. Diacetyl-HDA was present in untreated urine at 0.015–0.060 μg/l. Using base hydrolysis, the concentration range of monoacetyl-HDA in urine was 0.19–2.2 μg/l, 60-fold higher than in the untreated samples on average. HDA was detected only in one sample after base hydrolysis (0.026 μg/l). In contrast, acid hydrolysis yielded HDA concentrations ranging from 0.36 to 10.1 μg/l in these three samples. These findings demonstrate HDI metabolism via N-acetylation metabolic pathway and protein adduct formation resulting from occupational exposure to HDI.     

Analysis of dialkyl phosphate metabolites in hair using gas chromatography-mass spectrometry: A biomarker of chronic exposure to organophosphate pesticides

The aim of our study was to develop and validate an analytical approach for the quantitative determination of three dialkyl phosphate (DAP) metabolites, dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP) and diethyl phosphate (DEP), of organophosphate pesticides (OPs) in hair samples. The proposed methodology comprises a decontamination step, solid–liquid extraction, followed by liquid–liquid extraction, pentafluorobenzyl bromide derivatization, clean-up on Florisil/PSA column and analysis by gas chromatography–mass spectrometry (GC-MS). Extraction recovery, obtained from 50?mg hair samples spiked at two concentration levels, ranged from 56.1 to 107.9% and the within-day precision ranged from 13.5 to 17.5%. Limits of detection (LODs) ranged from 0.02 to 0.10?ng mg^?1. The results obtained from the analysis of hair samples of 30 agricultural workers show the suitability of the proposed method for monitoring people occupationally exposed to OPs. The most frequently detected compound was DEP followed by DMP. This is the first report on the detection of dialkyl phosphates in human hair which reflects the ability of hair testing to assess chronic exposure to OPs.     

Degradation of O,O-Dimethyl Phosphorodithioate by Thiobacillus thioparus TK-1 and Pseudomonas AK-2

O,O-Dimethyl phosphorodithioate (DMDTP) is an initial breakdown product of organophosphorus pesticides in fields. DMDTP is also released to natural environments by pesticide manufacturers. DMDTP-degrading microorganisms were not known. We isolated two bacteria from activated sludge. One of them, strain TK-1 identified as Thiobacillus thioparus, utilized DMDTP as a sole energy source and produced dimethyl phosphate (DMP) and sulfate. The other, strain AK-2 identified as Pseudomonas sp., utilized DMP as a sole energy and carbon source and degraded DMP to inorganic orthophosphate (P_i). DMDTP was degraded to P_i by the coaction of the two bacteria.     

Measurement of phenol and p-cresol in urine and feces using vacuum microdistillation and high-performance liquid chromatography

In this article, we describe a simple, sensitive, accurate, and repeatable method for the measurement of phenol and p-cresol (4-methylphenol) in human urine and feces. We examined a number of parameters to identify an optimal extraction protocol. Purification of sample extracts was achieved by low-temperature vacuum microdistillation. Separation was achieved in approximately 15 min by high-performance liquid chromatography (HPLC) with quantification by fluorescence at 284/310 nm. Limits of detection for phenol were 2 ng/ml for urine and 20 ng/g for feces, and those for p-cresol were 10 ng/ml for urine and 100 ng/g for feces. For comparison, approximate mean values for urine are 3 μg/ml for phenol and 30 μg/ml for p-cresol, and those for feces are 1 μg/g for phenol and 50 μg/g for p-cresol. An experienced analyst can process 60 samples each day using this method.     

Determination of ethylenethiourea in urine by liquid chromatography–atmospheric pressure chemical ionisation–mass spectrometry for monitoring background levels in the general population

This study reports a sensitive analytical method suitable for the quantitative analysis of ethylenethiourea (ETU) in human urine and its application to samples from the general population. Sample preparation involved the use of diatomaceous earth extraction columns to remove matrix interferences. Quantification was achieved by liquid chromatography–mass spectrometry using positive ion atmospheric pressure chemical ionisation. Within-day and between-day variability of 14% (n = 10) and 11% (n = 6), respectively, were obtained at 98 nmol/l (10 μg l⁻¹). The assay was linear over the investigated range 2.5–245 nmol/l, with a limit of detection of 2.5 nmol/l. The method was applied to monitoring background levels of ETU in urine samples from the general population in the UK. Results obtained from 361 spot samples contained ETU levels ranging from less than the detection limit (54% of samples) to a maximum of 15.8 μmol/mol creatinine (14.3 μg/g creatinine). The 95th percentile was 5.7 μmol/mol creatinine (5.2 μg/g creatinine).     

Solid-phase extraction of tramadol from plasma and urine samples using a novel water-compatible molecularly imprinted polymer

In this study, a novel method is described for the determination of tramadol in biological fluids using molecularly imprinted solid-phase extraction (MISPE) as the sample clean-up technique combined with high-performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and tramadol as template molecule. The novel imprinted polymer was used as a solid-phase extraction (SPE) sorbent for the extraction of tramadol from human plasma and urine. Various parameters affecting the extraction efficiency of the polymer have been evaluated. The optimal conditions for the MIP cartridges were studied. The MIP selectivity was evaluated by checking several substances with similar molecular structures to that of tramadol. The limit of detection (LOD) and limit of quantification (LOQ) for tramadol in urine samples were 1.2 and 3.5 μg L⁻¹, respectively. These limits for tramadol in plasma samples were 3.0 and 8.5 μg L⁻¹, respectively. The recoveries for plasma and urine samples were higher than 91%.     

Determination of chlorpyrifos metabolites in human urine by reversed-phase/weak anion exchange liquid chromatography-electrospray ionisation-tandem mass spectrometry

A liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of major chlorpyrifos (CP) metabolites, i.e. diethyl thiophosphate (DETP), diethyl phosphate (DEP), and 3,5,6-trichloro-2-pyridinol (TCP), in human urine was developed. Simultaneous separation of the parent compound and its primary biotransformation products was achieved within 20 min in gradient elution mode employing a mixed-mode reversed-phase/weak anion exchange (RP/WAX) separation principle. The analytical method was developed for a toxicokinetic study of an acute poisoning incidence with a CP containing pesticide formulation. An initial mass spectrometric screening performed with unprocessed urine samples revealed that CP is not excreted unchanged by the kidney. Hence, the quantitative assay was validated for DETP (quantifier transition: m/z 169-->95, qualifier transition: m/z 169-->141), DEP (m/z 153-->79, 153-->125), and TCP (m/z 196-->35, 198-->35) taking dibutyl phosphate (DBP) (m/z 209-->79, 209-->153) as internal standard. Clean-up of urine samples prior to LC-ESI-MS/MS analysis was carried out by a liquid-liquid extraction step with a mixture of ethylacetate and acetonitrile (70:30; v/v). Linearity was observed between 0.25 and 75 mgL(-1), and the signal-to-noise ratio at 0.25 mgL(-1) was better than six for the individual analytes. Recoveries, precision, and accuracies were all adequate across the validated range of 1-75 mgL(-1) for the present toxicological case study.     

Alkyl-methylimidazolium ionic liquids affect the growth and fermentative metabolism of Clostridium sp

In this study, the effect of ionic liquids, 1-ethyl-3-methylimidazolium acetate [EMIM][Ac], 1-ethyl-3-methylimidazolium diethylphosphate [EMIM][DEP], and 1-methyl-3-methylimidazolium dimethylphosphate [MMIM][DMP] on the growth and glucose fermentation of Clostridium sp. was investigated. Among the three ionic liquids tested, [MMIM][DMP] was found to be least toxic. Growth of Clostridium sp. was not inhibited up to 2.5, 4 and 4 g L⁻¹ of [EMIM][Ac], [EMIM][DEP] and [MMIM][DMP], respectively. [EMIM][Ac] at <2.5 g L⁻¹, showed hormetic effect and stimulated the growth and fermentation by modulating medium pH. Total organic acid production increased in the presence of 2.5 and 2 g L⁻¹ of [EMIM][Ac] and [MMIM][DMP]. Ionic liquids had no significant influence on alcohol production at <2.5 g L⁻¹. Total gas production was affected by ILs at ?2.5 g L⁻¹ and varied with type of methylimidazolium IL. Overall, the results show that the growth and fermentative metabolism of Clostridium sp. is not impacted by ILs at concentrations below 2.5 g L⁻¹.     

An ultrasensitive gold nanoparticles improved real-time immuno-PCR assay for detecting diethyl phthalate in foodstuff samples

A specific polyclonal antibody targeting diethyl phthalate (DEP) with the higher antibody titer at 1:120,000 has been obtained, and an ultrasensitive and high-throughput direct competitive gold nanoparticles improved real-time immuno-PCR (GNP–rt–IPCR) technique has been developed for detecting DEP in foodstuff samples. Under optimal conditions, a rather low linearity is achieved within a range of 4 pg L⁻¹ to 40 ng L⁻¹, and the limit of detection (LOD) is 1.06 pg L⁻¹. Otherwise, the GNP–rt–IPCR technique is highly selective, with low cross-reactivity values for DEP analogs (<5%). Finally, the concentrations of DEP in foodstuff samples by the GNP–rt–IPCR method range from 0.48 to 41.88 μg kg⁻¹. Satisfactory recoveries (88.39–112.79%) and coefficient of variation values (8.38–12.77%) are obtained. The consistency between the results obtained from GNP–rt–IPCR and gas chromatography–mass spectrometry (GC–MS) is 98.3%, which further proves that GNP–rt–IPCR is an accurate, reliable, rapid, ultrasensitive, and high-throughput method for batch determination of trace amounts of DEP in foodstuff samples.     

Liquid chromatography-tandem mass spectrometry analysis of urinary fluticasone propionate-17beta-carboxylic acid for monitoring compliance with inhaled-fluticasone propionate therapy

Background

Inhaled corticosteroids including fluticasone propionate (FP) are the most effective treatment for persistent-asthma. Noncompliance ranging from 20% to 80% of treated patients is associated with substantial health care costs, morbidity and fatalities. A noninvasive test to assess FP treatment compliance is needed. The major metabolite of FP is FP-17beta-carboxylic acid (FP17βCA) and is excreted in urine. This study demonstrates the development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure FP17βCA in urine and evaluation of FP17βCA urinary elimination.

Experimental

Fluorometholone was used as the internal standard. After acetonitrile precipitation, samples were extracted with dichloromethane, washed and dried. Reconstituted extract (60 μL) was subjected to reversed-phase chromatography and positive-ion mode LC-MS/MS analysis. Assay precision, linearity, recovery and sample stability were determined. Elimination evaluation included measurement of FP17βCA in urine collected daily from human subjects before (day 1), during treatment (days 2-5; dose FP-110 μg 2 puffs/day), and following cessation of FP therapy (days 6-14; n = 4).

Results

Linear range of the FP17βCA assay was 10.3-9510 pg/mL. Limit of quantitation (LOQ) was 10.3 pg/mL and recovery ranged from 85.8% to 111.9%. Inter-assay CVs were 7.4-12.0% for FP17βCA concentrations of 11.1-5117 pg/mL. Urine FP17βCA was absent in subjects prior to FP therapy, detectable (180-1991 ng FP17βCA/g creatinine) throughout the dosing period and reached below the LOQ at 6 days after therapy cessation.

Conclusions

Measurement of FP17βCA by LC-MS/MS has acceptable analytical performance for clinical use. These data support the clinical utility of measuring FP17βCA in urine to monitor patient compliance with FP therapy.     

Quantification of corticosteroids in bovine urine using selective solid phase extraction and reversed-phase liquid chromatography/tandem mass spectrometry

This paper presents the development of a simple liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to determine corticosteroids in bovine urine sample matrices. This method uses a single phase extraction (SPE) for cleaning of the sample with an Oasis MAX cartridge at pH 9.0–9.5 and elution by a neutral organic solvent (acetonitrile/dichloromethane), followed by separation on a GEMINI C18 column in the gradient mode with acetate buffer (pH 4.1)/methanol. A triple quadrupole mass spectrometer equipped with a multimode ion source, set to negative atmospheric pressure chemical ionization (APCI) in the multiple reaction monitoring mode was used for detection. The main advantage of this method over other commonly used methods includes the use of SPE with a low volume cartridge for sample preparation and no ion suppression effects from matrix components of the urine samples in the LC–MS/MS analysis. This allowed a reduction the quantification limits (decision limits, CCα) for the first time to 0.1 μg/L (1 and 0.2 μg/L for triamcinolone and flumethasone, respectively). The developed method was validated in accordance with the European Union Commission Decision 2002/657 EC. The recoveries and within-laboratory reproducibility varied from 77% to 115% and 87% to 107.5%, respectively, at 2, 3, and 4 μg/L levels of corticosteroids. The relative standard deviation (RSD) of the measurements was lower than 30%. The decision limit was calculated by multiplying the signal-to-noise ratio by 3 and the obtained values were in the range of 0.1–1.0 μg/L, confirmed by the analysis of twenty blank samples, which were spiked at the desired concentrations. The detection capability was calculated by the addition of the decision limit and the standard deviation followed by multiplication by 1.64 of the within-laboratory reproducibility at 2 μg/L of corticosteroids. The method was applied to four urine samples, giving concentrations of prednisolone (PRED) residues in the range from 0.3 to 0.9 μg/L.