GC–MS method for the determination of paraben preservatives in the human breast cancerous tissue

The general presumption that the preservative laden personal care products may be one of the causative agents for breast cancer, has remained a matter of controversy during this decade. Extensive studies have not been carried out to either prove or disprove the role of preservatives in breast cancer incidences. In this study we have developed a new method for the identification and quantification of the preservatives such as methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP) and butyl paraben (BuP) in breast tissue using Gas Chromatography and Mass Spectrometry (GC–MS). Tissue was extracted by using acetone:n-hexane mixture (1:1 v/v) and derivatized with N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). The extent of reaction time and the amount of MSTFA to attain greater derivatization were optimized. The developed method yielded good recovery (mean ± SD) of 99.8 ± 5.1, 96 ± 4.4, 107 ± 17 and 113 ± 13% with relative standard deviations (RSDs) of 5.1, 4.6, 15.6 and 13%, and the limits of detection (LOD) of 2.02, 1.05, 1.71 and 3.75 ng g^− 1 for MeP, EtP, PrP and BuP, respectively. The method was successfully validated for the determination of parabens including butyl paraben (log K_ow = 3.57) in cancerous breast tissues; this could be a promising one for screening of breast tissues and also the environment for paraben residues. As far as our knowledge goes this is the first GC–MS method for the determination of parabens in human tissue.     

Simultaneous determination of parabens,triclosan and triclocarban in water by liquid chromatography/electrospray ionisation tandem mass spectrometry

A method for the determination of several household biocides in water by liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI‐MS/MS) is presented. It permits the simultaneous determination of triclosan (TCS), triclocarban (TCC) and seven parabens, including the distinction between branched and linear isomers of propyl (i‐PrP and n‐PrP) and butyl parabens (i‐BuP and n‐BuP). Prior to LC/MS/MS, analytes are preconcentrated by solid‐phase extraction (SPE) on Oasis HLB (60 mg) cartridges at natural sample pH and subsequently eluted with 4 mL of methanol. This simple SPE procedure provides extraction recoveries above 85% except for raw wastewater, where it falls to 65% for TCC. The performance of the method was tested with two triple‐quadrupole LC/MS instruments from a low/mid and mid/high market range: a Varian 1200L and an API‐4000. The latter system provided between 3 and 80 times lower limits of quantification (LOQs) than the first one, in the 0.08–0.44 ng/L range for surface water. Moreover, a comparison of matrix effects on both instruments showed a very different behaviour, particularly in the case of parabens. For these compounds signal suppression was observed in the 1200L instrument and signal enhancement with the 4000 instrument. As a result, different calibration approaches were chosen for them and this pointed to the need of matrix effect re‐evaluation in method transfer between different LC/MS systems. The application of the method to real samples showed the ubiquity of methyl paraben (MeP) and n‐PrP (at the 1–6 µg/L in raw wastewater) and the coexistence of i‐BuP and n‐BuP at similar levels (ca. 100–200 ng/L in raw wastewater). Copyright © 2009 John Wiley & Sons, Ltd.     

Synthetic approaches to parabens molecularly imprinted polymers and their applications to the solid-phase extraction of river water samples

In this paper we describe the synthesis, characterisation and use of two distinct molecularly imprinted polymers (MIPs) prepared using esters of p-hydroxybenzoic acid (parabens) as templates: one MIP was synthesised by precipitation polymerisation using a semi-covalent molecularly imprinting strategy with methyl paraben as the template/target (MIP 1); the second MIP was prepared in monolithic form through a conventional non-covalent molecular imprinting strategy, with butyl paraben as the template (MIP 2). MIP 1 recognized methyl paraben, showed cross-selectivity for other parabens analytes used in the study and higher affinity towards these compounds than did a non-imprinted control polymer. Similarly, MIP 2 demonstrated higher affinity towards paraben analytes than a non-imprinted control polymer.For the analysis of environmental water samples, a solid-phase extraction (SPE) protocol was developed using MIP 2 as sorbent, and results were compared to a SPE using a commercial sorbent (Oasis HLB). With MIP 2 as sorbent and butyl paraben as target, when percolating 500 mL of river water spiked at 1 μg L⁻¹ through the SPE cartridge, and using 1 mL of isopropanol as cleaning solvent, a higher recovery of butyl 4-hydroxybenzoate (butyl paraben) and a cleaner chromatogram where achievable when using the MIP compared to the commercial sorbent.     

Single‐drop microextraction followed by in‐syringe derivatization and GC‐MS detection for the determination of parabens in water and cosmetic products

A single‐drop microextraction (SDME) method followed by in‐syringe derivatization and GC‐MS determination has been developed for analysis of five parabens, including methyl, ethyl, isopropyl, n‐propyl and n‐butyl paraben in water samples and cosmetic products. N,O‐Bis(trimethylsilyl)acetamide (BSA) was used as derivatization reagent. Derivatization reaction was performed inside the syringe barrel using 0.4 μL of BSA. Parameters that affect the derivatization yield such as temperature and time of the reaction were studied. In addition, experimental SDME parameters such as selection of organic solvent, addition of salt, extraction time and extraction temperature were investigated and optimized. The RSD of the method for aqueous samples varied from 8.1 to 13%. The LODs ranged from 0.001 (n‐butyl paraben) to 0.015 (methyl paraben) μg/L, and the enrichment factors were between 23 and 150.     

Comparative analysis of odorous volatile organic compounds between direct injection and solid-phase microextraction: Development and validation of a gas chromatography–mass spectrometry-based methodology

In this study, the feasibility of GC–MS was evaluated for the quantification of odorous volatile organic compounds (VOCs) in environmental samples. These included methyl ethyl ketone, isobutyl alcohol, methyl isobutyl ketone, and butyl acetate plus benzene, toluene, and xylene (BTX). For this purpose, the gaseous standard for these VOCs were analyzed by GC–MS with the aid of both direct injection (DI) into the GC injector and solid-phase microextraction (SPME). The liquid phase standard prepared independently was tested additionally by the DI method as a reference to gaseous calibration. The detection limit (DL) values, when tested for basic quality assurance in this study, showed large differences between DI (0.002–0.007 ng) and SPME method (1.03–1.81 ng) in terms of absolute mass. The DL values, when expressed in terms of concentration (v/v), showed considerable improvement in SPME (below 0.40 nmol mol⁻¹) relative to the DI method (∼6–15 nmol mol⁻¹). The reliability of the GC–MS method was further validated through an analysis of real environmental samples collected from an industrial area.     

Ultrasound‐assisted temperature‐controlled ionic liquid emulsification microextraction coupled with capillary electrophoresis for the determination of parabens in personal care products

We developed a CE and ultrasound‐assisted temperature‐controlled ionic liquid emulsification microextraction method for the determination of four parabens (methyl paraben, ethyl paraben, propyl paraben, and butyl paraben) in personal care products including mouthwash and toning lotion. In the proposed extraction procedure, ionic liquid (IL, 1‐octyl‐3‐methylimidazolium hexafluorophosphate) was used as extraction solvent, moreover, no disperser solvent was needed. Parameters affecting the extraction efficiency including volume of IL, heating temperature, ultrasonic time, extraction time, sample pH, ionic strength, and centrifugation time were optimized. Under the optimized conditions, the method was found to be linear over the range of 3–500 ng/mL with coefficient of determination (R²) in the range of 0.9990–0.9998. The LODs and LOQs for the four parabens were 0.45–0.72 ng/mL and 1.50–2.40 ng/mL, respectively. Intraday and interday precisions (RSDs, n = 5) were in the range of 5.4–6.8% and 7.0–8.7%, respectively. The recoveries of parabens at different spiked levels ranged from 71.9 to 119.2% with RSDs less than 9.5%.     

Gas chromatography–mass spectrometry screening methods for select UV filters,synthetic musks,alkylphenols, an antimicrobial agent,and an insect repellent in fish

Two screening methods have been developed for simultaneous determination of ten extensively used personal care products (PCPs) and two alkylphenol surfactants in fish. The methods consisted of extraction, clean-up, derivatization and analysis by gas chromatography–mass spectrometry with selected ion monitoring (GC–SIM–MS) or gas chromatography–tandem mass spectrometry (GC–MS/MS) techniques. Among solvents tested to assess recovery of target compounds from 1-g tissue homogenates, acetone was selected as optimal for extracting compounds with dissimilar physicochemical properties from fish tissue. Initial experiments confirmed that GC–SIM–MS could be applied for analysis of lean fillet tissue (<1% lipid) without gel-permeation chromatography (GPC), and this approach was applied to assess the presence of target analytes in fish fillets collected from a regional effluent-dominated stream in Texas, USA. Benzophenone, galaxolide, tonalide, and triclosan were detected in 11 of 11 environmental samples at concentrations ranging from; 37 to 90, 234 to 970, 26 to 97, and 17 to 31 ng/g, respectively. However, performance of this analytical approach declined appreciably with increasing lipid content of analyzed tissues. Successful analysis of samples with increased lipid content was enabled by adding GPC to the sample preparation protocol and monitoring analytes with tandem mass spectrometry. Both analytical approaches were validated using fortified fillet tissue collected from locations expected to be minimally impacted by anthropogenic influences. Average analyte recoveries ranged from 87% to 114% with RSDs <11% and from 54% to 107% with RSDs <20% for fish tissue containing <1% and 4.9% lipid, respectively. Statistically derived method detection limits (MDLs) for GC–SIM–MS and GC–MS/MS methodologies ranged from 2.4 to 16 ng/g, and 5.1 to 397 ng/g, respectively.     

固相萃取-气相色谱/质谱法同时测定化妆品中的邻苯二甲酸酯和对羟基苯甲酸酯

采用超声协助甲醇提取、固相萃取净化、气相色谱/选择离子质谱联用法,同时测定化妆品中8种邻苯二甲酸酯和4种对羟基苯甲酸酯。该方法线性范围广、重现性好、快速简便、干扰小。样品的加标回收率为80%～100%;含量检测的相对标准偏差小于10%;方法的检出限为0.1~5.0 μg/kg。用该方法对15种实际样品中的12种残留物进行定量检测,结果表明除了一种样品中不含待测物外,其余样品均检测到3~7种待测物。其中以对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、邻苯二甲酸丙酯、邻苯二甲酸环己酯和邻苯二甲酸乙基庚基酯为主。     

Analytical performance of a triple quadrupole mass spectrometer compared to a high resolution mass spectrometer for the analysis of polybrominated diphenyl ethers in fish

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardants used globally in many consumer products and industrial applications. Traditionally, gas chromatography–high resolution mass spectrometry (GC–HR-MS) is the method of choice for analysis of PBDEs in environmental samples because it offers high sensitivity and selectivity, resulting in less interferences. However, the specificity offered by gas chromatography-triple quadrupole tandem mass spectrometry (GC–QQQ-MS/MS), operated in selected reaction monitoring mode, provides a more affordable alternative to GC–HR-MS for the analysis of PBDEs in complex environmental samples. In this study, an analytical method was developed for the analysis of 41 PBDE congeners in fish using GC–QQQ-MS/MS. Results from the analysis of three fish species [lake trout (Salvelinus namaycush), yellow perch (Perca flavescens), and round goby (Neogobius melanostomus)] using GC–QQQ-MS/MS were compared with those obtained by GC–HR-MS. These species were selected because they represent varying levels of lipid-rich matrix and contaminant loads. Instrumental limits of detection for the GC–QQQ-MS/MS ranged from 0.04 pg to 41 pg, whereas those for the GC–HR-MS ranged from 5 pg to 85 pg. The PBDE values obtained from these two methods were highly correlated, R² values >0.7, for all three fish species, supporting the suitability of GC–QQQ-MS/MS for analysis of PBDEs in fish with varying fat content.     

Optimisation of a headspace-solid-phase micro-extraction method for simultaneous determination of organometallic compounds of mercury,lead and tin in water by gas chromatography–tandem mass spectrometry

In this work, a headspace-solid-phase micro-extraction (HS-SPME) combined with gas chromatography–mass spectrometry (GC–MS) method for multielemental speciation of organometallic compounds of mercury, lead and tin in water samples was upgraded by the introduction of tandem mass spectrometry (MS/MS) as detection technique. The analytical method is based on the ethylation with NaBEt₄ and simultaneous headspace-solid-phase micro-extraction of the derivative compounds followed by GC–MS/MS analysis. The main experimental parameters influencing the extraction efficiency such as derivatisation time, extraction time and extraction temperature were optimized. The overall optimum extraction conditions were the following: a 50 μm/30 μm divinyl-benzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) SPME fibre, 150 min derivatisation time, 15 min extraction time, sample agitation at 250 rpm and 40 °C extraction temperature. The analytical characteristics of the HS-SPME method combined with GC–MS and GC–MS/MS were evaluated. The combination of both techniques HS-SPME and GC–MS/MS allowed to attain lower limits of detection (4–33 ng l⁻¹) than those obtained by HS-SPME–GC–MS (17–45 ng l⁻¹). The proposed method presented good linear regression coefficients (r² > 0.9970) and repeatability (4.8–21.0%) for all the compounds under study. The accuracy of the method measured as the average percentage recovery of the compounds in spiked river water and seawater samples was higher than 80% for all the compounds studied, except for monobutyltin in the river water sample. A study of the uncertainty associated with the analytical results was also carried out.     

Inclusion complexes of <Emphasis Type="Italic">p</Emphasis>-hydroxybenzoic acid esters and γ-cyclodextrin

The alkyl esters of p-hydroxybenzoic acid (parabens) are commonly used as preservatives in cosmetics, food products and pharmaceutical formulations because of their wide range antimicrobial activity. However, the usage of parabens in aqueous media has been hampered, especially parabens with long alkyl chains, due to their low aqueous solubility. One approach to increase their solubility is cyclodextrins (CDs) complexation to form water-soluble inclusion complexes. γCD has the widest hydrophobic central cavity and the highest water solubility among natural CDs. Hence, inclusion complexes between γCD and parabens of various alkyl chain lengths were investigated. Results from phase-solubility studies show that methyl- and ethylparaben form various complexes of paraben/γCD (i.e. 1:1, 2:1, etc.) while the 1:1 complex was dominant in propyl- and butylparaben/γCD complex solution. Moreover, the effect of the paraben complexation on the critical aggregation concentration (cac) of γCD in aqueous solutions was determined. It was found that the longer the paraben alkyl chain was the more influence it had on the γCD cac. In pharmaceutical formulations the mixture of parabens (i.e. binary, ternary and quaternary) has been used to maximize antimicrobial effect. It is important to determine how mixtures of parabens affect the solubility of γCD and its cac values upon formation of inclusion complexes. Competition of the different parabens for a space in the γCD central cavity was evaluated by comparing the γCD cac values obtained in presence of the individual parabens and their mixtures.     

High-throughput approach for analysis of multicomponent gas chromatographic–mass spectrometric signals

Hyphenated techniques such as gas chromatography–mass spectrometry (GC–MS) or high-performance liquid chromatography–mass spectrometry (LC–MS) produce a large amount of data in a form of two-way data matrix. It has been a great challenge to furthest extract the useful information from the data. In this work, a chemometric approach based on a modification of adaptive immune algorithm (AIA) was proposed for a high-throughput analysis of the multicomponent overlapping GC–MS signals. With the proposed method, the chromatographic profile of each component in an overlapping signal can be extracted independently and sequentially along the retention time. In order to show the efficiency of the method, a stimulated GC–MS data of six components with background and an experimental GC–MS data of 40 pesticides were investigated. It was found that the multicomponent overlapping GC–MS signals could be fast and accurately resolved. Furthermore, the quantitative property of the extracted information was also investigated. The correlation coefficients (r) between the peak area and the added volumes of the sample are in the range 0.9658–0.9953.     

Molecularly imprinted polymer for the extraction of parabens from environmental solid samples prior to their determination by high performance liquid chromatography-ultraviolet detection

An analytical methodology incorporating a molecularly imprinted solid-phase extraction procedure (MISPE) has been developed for the determination of parabens in environmental solid samples. Four different polymers were prepared combining the use of acetonitrile or toluene as porogen, and 4-vinylpyridine (VP) or methacrylic acid (MAA) as monomer, using benzylparaben (BzP) as a template molecule. Although all the polymers were able to recognize the template in rebinding experiments, the MIP prepared in toluene using MAA showed better performance. This polymer was also capable of recognizing other parabens (methyl, ethyl, isopropyl, propyl, isobutyl, butyl and benzylparaben) allowing to develop an appropriated MISPE procedure for this family of compounds. The extraction of the parabens from environmental solid samples was performed by ultrasonic assisted extraction in small columns (SAESC), and this procedure next to MISPE as clean-up step followed by HPLC-UV determination was successfully used for the determination of parabens in soil and sediment samples of different locations. Recoveries ranging from 80% to 90% have been achieved depending on the compound and the samples, and limits of detection (LODs) were under 1 ng g⁻¹ for all the compounds, making this method suitable for the determination of parabens in environmental solid matrices. The method was further applied to the determination of paraben contents in real samples, founding levels up to 11.5 ng g⁻¹ in sea sediments.     

Comparative study for the analysis of parabens by micellar electrokinetic capillary chromatography with and without large-volume sample stacking technique

The separation and determination of four parabens (methyl, ethyl, propyl, and butyl p-hydroxybenzoate) which are commonly used as preservatives in cosmetic products, by micellar electrokinetic capillary chromatography (MEKC) with and without large-volume sample stacking (LVSS) technique were compared. As an effective on-line concentration technique, LVSS was successfully combined with MEKC to determine neutral parabens in an acidic media. The effects of some typical parameters such as sample volume, buffer pH, temperature, and concentration of surfactant were examined. The detection limits for this LVSS-MEKC method were found to be 3.0 × 10⁻⁷ M for each of the parabens based on the signal-to-noise ratio of 3, which were around 300 times lower than normal MEKC technique. The curves of peak response versus concentration were linear from 1.0 × 10⁻⁶ to 5.0 × 10⁻⁵ M with regression coefficients of 0.9987, 0.9960, 0.9925 and 0.9864, respectively. A simple and easy-manipulative sample preparation method was developed and validated by analyzing commercially available cosmetic samples. It was found that with current sample preparation process and instrumentation system, 0.5 g of sample is enough for the analysis of parabens preservatives in cosmetic product with satisfactory results.     

Selective and quantitative analysis of 4-hydroxybenzoate preservatives by microemulsion electrokinetic chromatography

A microemulsion electrokinetic chromatography (MEEKC) method has been developed and validated for the determination of 4-hydroxybenzoates and their impurities. These materials are commonly known as parabens and are widely used as preservatives in foods, cosmetics and pharmaceuticals. The method was shown to be selective and quantitative for the methyl, ethyl, propyl and butyl esters of 4-hydroxybenzoic acid. An internal standard, 4-hydroxyacetophenone, was employed to improve injection precision and detector linearity. In addition, 4-hydroxybenzoic acid, the major degradent, could also be monitored at the 0.1% (m/m) level. The method was successfully validated for assay and detection of the impurities in 4-hydroxybenzoic acid methyl ester and 4-hydroxybenzoic acid propyl ester samples and for the determination of 4-hydroxybenzoic acid methyl ester in a liquid pharmaceutical formulation. The determination of paraben content by MEEKC in a liquid sample was consistent with HPLC analysis. This work is the first reported validated MEEKC method and shows that the methodology can be successfully implemented into routine quality control testing.     

A fast method for the identification of Mycobacterium tuberculosis in sputum and cultures based on thermally assisted hydrolysis and methylation followed by gas chromatography–mass spectrometry

A fast gas chromatography–mass spectrometry (GC–MS) method with minimum sample preparation is described for early diagnosis of tuberculosis (TB). The automated procedure is based on the injection of sputum samples which are then methylated inside the GC injector using thermally assisted hydrolysis and methylation (THM). The THM–GC–MS procedure was optimized for the injection of sputum samples. For the identification of Mycobacterium tuberculosis the known marker tuberculostearic acid (TBSA) and other potential markers were evaluated. Hexacosanoic acid in combination with TBSA was found to be specific for the presence of M. tuberculosis. For validation of the method several sputum samples with different viscosities spiked with bacterial cultures were analyzed. Finally, 18 stored sputum samples collected in Vietnam from patients suspected to suffer from TB were re-analyzed in Amsterdam by microscopy after decontamination/concentration and using the new THM–GC–MS method. No false positives were found by THM–GC–MS and all patients who were diagnosed with TB were also found positive using our newly developed THM–GC–MS method. These results show that the new fast and sensitive THM–GC–MS method holds great potential for the diagnosis of TB.     

Automated on-line column-switching HPLC-MS/MS method with peak focusing for measuring parabens, triclosan, and other environmental phenols in human milk

Parabens (esters of p-hydroxybenzoic acid) and triclosan are widely used as preservatives and antimicrobial agents, respectively, in personal care products, pharmaceuticals, and food processing. Because of their widespread use and potential risk to human health, assessing human exposure to these compounds in breastfed infants is of interest. We developed a sensitive method, using a unique on-line solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry system with peak focusing feature, to measure in human milk the concentrations of five parabens (methyl-, ethyl-, propyl-, butyl-, and benzyl parabens), triclosan, and six other environmental phenols: bisphenol A (BPA); ortho-phenylphenol (OPP); 2,4-dichlorophenol; 2,5-dichlorophenol; 2,4,5-trichlorophenol; and 2-hydroxy-4-methoxybenzophenone (BP-3). The method, validated by use of breast milk pooled samples, shows good reproducibility (inter-day coefficient of variations ranging from 3.5% to 16.3%) and accuracy (spiked recoveries ranging from 84% to 119% at four spiking levels). The detection limits for most of the analytes are below 1 ng mL(-1) in 100 microL of milk. We tested the usefulness of the method by measuring the concentrations of these twelve compounds in four human milk samples. We detected methyl paraben, propyl paraben, triclosan, BPA, OPP, and BP-3 in some of the samples tested. The free species of these compounds appear to be the most prevalent in milk. Nevertheless, to demonstrate the utility of these measures for exposure and risk assessment purposes, additional data about sampling and storage of the milk, and on the stability of the analytes in milk, are needed.     

Development and validation of a single RP-HPLC assay method for analysis of bulk raw material batches of four parabens that are widely used as preservatives in pharmaceutical and cosmetic products

A stability-indicating, robust, fast, and user friendly reversed-phase high-performance liquid chromatographic (RP-HPLC) assay method has been developed and validated for the analysis of commercial raw material batches of methylparaben, ethylparaben, propylparaben, and butylparaben. These four parabens are widely used as preservatives in pharmaceutical and cosmetic products. Accurate assay value of each of the parabens in their respective commercial lots is critical to determine the correct weight of the paraben that is needed to obtain the target concentration of the paraben in a specific lot of pharmaceutical or cosmetic products. Currently, there are no single HPLC assay methods (validated as per ICH requirements) available in the literature that can be used to analyze the commercial lots of each of the four parabens. The analytical method reported herein analyzes all four parabens in less than 10 min. The method presented in this report was successfully validated as per ICH guidelines. Therefore, this method can be implemented in QC laboratories to analyze and assay the commercial bulk lots of the four parabens.     

Isocratic liquid chromatographic determination of three paraben preservatives in hygiene wipes using a reversed phase core-shell narrow-bore column

The first HPLC method for the separation of three paraben preservatives (methyl-, ethyl- and propyl parabens) using a core-shell analytical column is reported in this study. The separation was completed in less than 8 min at a low flow rate of 0.4 mL min⁻¹ and an isocratic mobile phase containing 20% acetonitrile as organic modifier. The backpressure was < 200 bar in all cases, enabling the usage of conventional HPLC equipment. The proposed analytical procedure was validated for linearity (0.5–20 μg L⁻¹), limits of detection (15–43 μg L⁻¹) and quantification (50–142 μg L⁻¹), selectivity, within day (1.3–1.5%) and day-to-day (3.4–4.6%) precision and accuracy. The proposed method has been applied to the determination of the selected paraben preservatives in commercially available hygiene wipes. The mean percent recoveries were found to be in the range of 98.0–98.4%.      

Issues pertaining to the analysis of buprenorphine and its metabolites by gas chromatography–mass spectrometry

“Substitution therapy” and the use of buprenorphine (B) as an agent for treating heroin addiction continue to gain acceptance and have recently been implemented in Taiwan. Mature and widely utilized gas chromatography–mass spectrometry (GC–MS) technology can complement the low cost and highly sensitive immunoassay (IA) approach to facilitate the implementation of analytical tasks supporting compliance monitoring and pharmacokinetic/pharmacogenetic studies. Issues critical to GC–MS analysis of B and norbuprenorphine (NB) (free and as glucuronides), including extraction, hydrolysis, derivatization, and quantitation approaches were studied, followed by comparing the resulting data against those derived from IA and two types of liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods. Commercial solid-phase extraction devices, highly effective for recovering all metabolites, may not be suitable for the analysis of free B and NB; acetyl-derivatization products exhibit the most favorable chromatographic, ion intensity, and cross-contribution characteristics for GC–MS analysis. Evaluation of IA, GC–MS, and LC–MS/MS data obtained in three laboratories has proven the 2-aliquot GC–MS protocol effective for the determination of free B and NB and their glucuronides.