Rapid extraction and preservation of genomic DNA from human samples

Simple and rapid extraction of human genomic DNA remains a bottleneck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab and saliva samples. DNA is attracted onto a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle four microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for 1 month was demonstrated for captured DNA, facilitating straightforward collection, delivery, and handling of genomic DNA in an environment-friendly protocol.

Optimization of DNA extraction from dental remains

Efficient DNA extraction procedures is a critical step involved in the process of successful DNA analysis of such samples. Various protocols have been devised for the genomic DNA extraction from human tissues and forensic stains, such as dental tissue that is the skeletal part that better preserves DNA over time. However DNA recovery is low and protocols require labor‐intensive and time‐consuming step prior to isolating genetic material. Herein, we describe an extremely fast procedure of DNA extraction from teeth compared to classical method. Sixteen teeth of 100‐year‐old human remains were divided into two groups of 8 teeth and we compared DNA yield, in term of quantity and quality, starting from two different sample preparation steps. Specifically, teeth of group 1 were treated with a classic technique based on several steps of pulverization and decalcification, while teeth of group 2 were processed following a new procedure to withdraw dental pulp. In the next phase, the samples of both group underwent the same procedure of extraction, quantification and DNA profile analysis. Our findings provide an alternative protocol to obtain a higher amount of good quality DNA in a fast time procedure, helpful for forensic and anthropological studies.     

On-line identification of depurinating DNA adducts in human urine by capillary electrophoresis--fluorescence line narrowing spectroscopy

The benzo[a]pyrene (BP)-derived 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) depurinating one-electron oxidation adduct was identified in the urine extracts of coal-smoke-exposed humans for the first time. Urine samples were prepared by solid-phase extraction and reversed-phase high-performance liquid chromatography. Subsequently, the BP-6-N7Gua adduct was identified on-line with capillary electrophoresis-- fluorescence line narrowing spectroscopy (CE-FLNS) at 4.2 K. The daily excretion of BP-6-N7Gua in human urine of individuals exposed to coal smoke was approximately 226 pmol per micromol of creatinine. Due to the high level of excretion we propose that BP-6-N7Gua adducts found in urine could serve as effective biomarkers for risk assessment of BP exposure. The results demonstrate that CE-FLNS allows for on-line separation and DNA adducts identification in complex fluid extracts.     

Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits

Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.     

Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes

An automatic DNA fragment collector using capillary array gel electrophoresis has been developed. A sheath flow technique is used for not only detection but also collection of DNA fragments. In a sheath flow cell, the DNA fragments separated by 16 capillaries flow independently into corresponding sampling capillaries. The fraction collector consists of 16 sampling trays and each sampling tray is set beneath each end of the sampling capillaries to collect the flow-through DNA fragments. Certain DNA fragments are automatically sorted by controlling the movement of the sampling trays according to the signals from the system. The collector experimentally separated two mixtures of polymerase chain reaction (PCR) products: one prepared by using eight different sizes (base lengths from 161 to 562) of DNAs; and the other prepared by a differential display (DD) method with cDNA fragments. Collected DNA fragments are amplified by PCR and measured by electrophoresis. DNA fragments with base length differences of one (base lengths 363 and 364) were successfully separated. A separated DNA fragment from the DD sample was also successfully sequenced. In addition, differentially expressed DNA fragments were automatically sorted by comparative analysis, in which two similar cDNA fragment groups, labeled by two different fluorophores, respectively, were analyzed in the same gel-filled capillary. These results show that the automatic DNA fragment collector is useful for gene hunting in research fields such as drug discovery and DNA diagnostics.     

Determination of free and total phenylacetic and p- and m-hydroxyphenylacetic acids in human urine by liquid chromatography with fluorimetric detection

A highly sensitive and rapid liquid chromatographic method for the determination of free and total phenylacetic and p- and m-hydroxyphenylacetic acids in human urine is described. After extraction of urine with diethyl ether, these acids and phenylpropionic acid (internal standard) are converted into the corresponding fluorescent derivatives by treatment with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated on a reversed-phase column (Radial-Pak cartridge C18) with aqueous 65% (v/v) methanol and detected fluorimetrically. The detection limits for phenylacetic and p- and m-hydroxyphenylacetic acids are 5, 30 and 100 fmol, respectively, at a signal-to-noise ratio of 5 in a 20-microliter injection volume. This sensitivity permits precise determination of the free and total acids in 20 microliter of normal human urine.     

High-performance isotope-labeling liquid chromatography mass spectrometry for investigating the effect of drinking Goji tea on urine metabolome profiling

Urine is a human biofluid that is widely used for metabolomics research on disease biomarker discovery.Ideally,the metabolome profiles generated from comparative groups of individuals should mainly consist of the endogenous human metabolites that reflect the healthy states of the individuals.However,external factors,such as diet,may alter the urine metabolome profile by either introducing a significant amount or variety of exogenous metabolites to urine or inducing changes of the metabolome profile.Thus,strict control of the external factors during the sample collection process is critical for urine metabolomics aimed at discovery of disease biomarkers.In this work,we describe a study to determine the effect of drinking Goji tea,which is considered a nutritional supplement drink in some regions of the world,on urine metabolome profile.The purpose of this work is not to determine the nutritional values of Goji tea,but to investigate whether drinking a moderate amount of Goji tea 1-3 h(short-term effect)or 12 h(longer-term effect)before urine collection can cause significant variations of urine metabolome profiles.A highly sensitive dansylation isotope labeling liquid chromatography mass spectrometry(LC-MS)method was used to determine the urine metabolomes before and after drinking Goji tea.From the studies of the short term(<3 h)and longer term(12 h)effects of drinking Goji tea,it is clear that the consumption of a moderate amount of Goji tea does not affect the urine metabolome significantly.Fasting for 12 h should be sufficient to remove any potential interference of Goji metabolites from the human urine metabolome profile.     

Solidified floating organic drop microextraction combined with high performance liquid chromatography for the determination of carbamazepine in human plasma and urine samples

Solidified floating organic drop microextraction (SFODME) in combination with high performance liquid chromatography was used for separation/preconcentration and determination of carbamazepine (CBZ) in human plasma and urine samples. Parameters that affect the extraction efficiency such as the type and volume of extraction solvent, ionic strength, sodium hydroxide concentration, stirring rate, sample volume and extraction time, were investigated and optimized. Under the optimum conditions (extraction solvent, 40 μL of 1-undecanol; sodium hydroxide concentration, 1 mol/L; temperature, 50 ℃; stirring speed, 400 r/min; sample volume, 8 mL; sodium chloride concentration, 3% (w/v) and extraction time, 60 min) the calibration curve was found to be linear in the mass concentration range of 0.4-700.0 μg/L. The limit of detection (LOD) was 0.1 μg/L and the relative standard deviation (RSD) for six replicate extraction and determination of carbamazepine at 100 μg/L level was found to be 4.1%. The method was successfully applied to the determination of CBZ in human plasma and urine samples.     

Label-Free DNA Biosensor for Electrochemical Detection of Short DNA Sequences Related to Human Papilloma Virus

Abstract

Highly sensitive label-free techniques of DNA determination are particularly interesting in relation to the present development of an electrochemical hybridization biosensor for the detection of short DNA fragments specific to the human papilloma virus (HPV). Unlabeled DNA probes have been immobilized by spontaneous coadsorption of thiolated single-stranded oligonucleotides (HS-ssDNA) onto the sensing surface of a screen-printed gold electrode (SPGE). The covalently immobilized single-stranded DNA probe (HS-ssDNA) could selectively hybridize with its complementary DNA (cDNA) in solution to form double-stranded DNA (dsDNA) on the surface. DNA is treated with acid (e.g., 0.5 M chloridric acid), and the acid-released purine bases are directly determined by square wave voltammetry (SWV).

Variables of the probe-immobilization and hybridization steps are optimized to offer convenient quantitation of HPV DNA target, in connection with a short hybridization time. Peak currents were found to increase in the following order: hybrid-modified SPGE, 11-base mismatched modified SPGE, 18-base mismatched SPGE, and the probe modified SPGE. Control experiments with noncomplementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. The effect of the target DNA concentration on the hybridization signal was also studied. Under optimal conditions, this sensor has a good calibration range with HPV DNA sequence detection limit of 2 pg · ml^?1 (S/N = 3).     

Direct visual detection of DNA based on the light scattering of silica nanoparticles on a human papillomavirus DNA chip

A detection system for a human papillomavirus (HPV) DNA chip based on the light scattering of aggregated silica nanoparticle probes is presented. In the assay, a target HPV DNA is sandwiched between the capture DNA immobilized on the chip and the probe DNA immobilized on the plain silica nanoparticle. The spot where the sandwich reaction occurs appears bright white and is readily distinguishable to the naked eye. Scanning electron microscopy images clearly show the aggregation of the silica nanoparticle probes. When three different sized (55 nm, 137 nm, 286 nm) plain silica nanoparticles were compared, probes of the larger silica nanoparticles showed a higher scattering intensity. Using 286-nm silica nanoparticles, the spots obtained with 200 pM of target DNA were visually detectable. The demonstrated capability to detect a disease related target DNA with direct visualization without using a complex detection instrument provides the prerequisite for the development of portable testing kits for genotyping.     

A high-throughput liquid chromatography/tandem mass spectrometry method for simultaneous quantification of a hydrophobic drug candidate and its hydrophilic metabolite in human urine with a fully automated liquid/liquid extraction

ABT-869 (A-741439) is an investigational new drug candidate under development by Abbott Laboratories. ABT-869 is hydrophobic, but is oxidized in the body to A-849529, a hydrophilic metabolite that includes both carboxyl and amino groups. Poor solubility of ABT-869 in aqueous matrix causes simultaneous analysis of both ABT-869 and its metabolite within the same extraction and injection to be extremely difficult in human urine. In this paper, a high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method has been developed and validated for high-speed simultaneous quantitation of the hydrophobic ABT-869 and its hydrophilic metabolite, A-849529, in human urine. The deuterated internal standards, A-741439D(4) and A-849529D(4), were used in this method. The disparate properties of the two analytes were mediated by treating samples with acetonitrile, adjusting pH with an extraction buffer, and optimizing the extraction solvent and mobile phase composition. For a 100 microL urine sample volume, the lower limit of quantitation was approximately 1 ng/mL for both ABT-869 and A-849529. The calibration curve was linear from 1.09 to 595.13 ng/mL for ABT-869, and 1.10 to 600.48 ng/mL for A-849529 (r2 > 0.9975 for both ABT-869 and A-849529). Because the method employs simultaneous quantification, high throughput is achieved despite the presence of both a hydrophobic analyte and its hydrophilic metabolite in human urine.     

Direct tandem mass spectrometry for the simultaneous assay of opioids,cocaine and metabolites in dried urine spots

A micro-analytical method based on spotting urine samples (20 μL) onto blood/urine spot collection cards followed by air-drying and extraction (dried urine spot, DUS) was developed and validated for the screening/confirmation assay of morphine, 6-methylacetylmorphine (6-MAM), codeine, cocaine and benzoylecgonine (BZE). Acetonitrile (3 mL) was found to be a useful solvent for target extraction from DUSs under an orbital-horizontal stirring at 180 rpm for 10 min. Determinations were performed by direct electrospray ionization tandem mass spectrometry (ESI-MS/MS) under positive electrospray ionization conditions, and by using multiple reaction monitoring (MRM) with one precursor ion/product ion transition for the identification and quantification (deuterated analogs of each target as internal standards) of each analyte. The limits of detection of the method were 0.26, 0.94, 1.5, 1.1, and 2.0 ng mL⁻¹, for cocaine, BZE, codeine, morphine and 6-MAM, respectively; whereas, relative standard deviations of intra- and inter-day precision were lower than 8 and 11%, respectively, and intra- and inter-day analytical recoveries ranged from 94 ± 4 to 105 ± 3%. The small volume of urine required (20 μL), combined with the simplicity of the analytical technique makes it a useful procedure for screening/quantifying drugs of abuse. The method was successfully applied to the analysis of urine from polydrug abusers.     

Comparison and evaluation of analysis procedures for the quantification of (2-methoxyethoxy)acetic acid in urine

Several extraction and derivatization procedures were evaluated for the quantification of (2-methoxyethoxy)acetic acid (MEAA) in urine. MEAA is a metabolite and a biomarker for exposure to 2-(2-methoxyethoxy)ethanol, a glycol ether with widespread use in various industrial applications and the specific use as an anti-icing additive in the military jet fuel formulation JP-8. Quantification of glycol ether biomarkers is an active area of analytical research. Various sample preparation procedures were evaluated: liquid–liquid extraction (LLE) using ethyl acetate yielded the highest recovery, and solid-phase extraction (SPE) gave low recovery of MEAA. Two derivatization procedures were thoroughly investigated and validated, namely, silylation of MEAA with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), and esterification of MEAA using ethanol. Quantification was performed by gas chromatography (GC) with a mass spectrometer as detector and using a polydimethylsiloxane (HP-1) capillary column. Deuterated 2-butoxyacetic acid (d-BAA) was used as an internal standard. Recovery studies of spiked human urine demonstrated the accuracy and precision of both procedures. The limit of detection (LOD) and other figures of merit for both derivatization procedures will be discussed in detail. Applications of these analysis procedures are also discussed. Disclaimers Mention of company names and/or products does not constitute endorsement by the Centers for Disease Control and Prevention (CDC). The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.     

Analysis of anaesthetics and analgesics in human urine by headspace SPME and GC

The determination of widely used anaesthetic and analgesic drugs in biological fluids is of major clinical importance. Typical methods used for sample preparation employ liquid–liquid extraction protocols which are complex, costly, not handy and not amenable to automation. In the present communication, we report the development of a methodology that employs headspace solid‐phase microextraction (HS‐SPME) for the determination of four anaesthetic (lidocaine, midazolam, diazepam and ketamine) and three analgesic drugs (fentanyl, remifentanyl and codeine) in human urine. Important parameters controlling SPME were studied: selection of SPME fibre, type and amount of salt added, preheating and extraction time, extraction temperature, sample volume and desorption time. GC with nitrogen phosphorus detection (GC‐NPD) facilitates sensitive and selective detection of the anaesthetics. The developed method renders an efficient tool for the precise and sensitive determination of the anaesthetics and analgesics in human urine (RSDs ranged from 7.7 to 12.6%, whereas LODs ranged from 0.01 to 1.5 ng/mL). The method was applied to the determination of the anaesthetics and analgesics in human urine from patients that had undergone coronary by‐pass surgery operations. The proposed protocol can function as an attractive alternative for clinical acute intoxications and medico‐legal cases.     

Determination of fipronil by solid-phase microextraction and gas chromatography-mass spectrometry.

A method for the determination of trace amounts of the insecticide fipronil was developed using solid-phase microextraction-gas chromatography-mass spectrometry and selected ion monitoring. Fipronil was extracted with a fused-silica fiber coated with 85 microm polyacrylate. The effects of pH, ionic strength, sample volume, extraction and desorption times as well as the extraction temperature were studied. Lindane was used as an internal standard. The linear concentration range of application was 0.3-100 ng ml(-1) of fipronil, with a relative standard deviation of 9.5% (for a level of 50 ng ml(-1)) and a detection limit of 0.08 ng ml(-1). The method was applied to check the eventual existence of fipronil above this limit in water and soil samples from Granada (Spain) as well as in human urine samples. The method validation was completed with spiked matrix samples. The method can be applied as a monitoring tool for water, soil and urine, in the investigation of environmental and occupational exposure to fipronil.     

Determination of famotidine in low-volume human plasma by normal-phase liquid chromatography/tandem mass spectrometry

A rapid, sensitive and robust assay procedure using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the determination of famotidine in human plasma and urine is described. Famotidine and the internal standard were isolated from plasma samples by cation-exchange solid-phase extraction with benzenesulfonic acid (SCX) cartridges. The urine assay used direct injection of a diluted urine sample. The chromatographic separation was accomplished by using a BDS Hypersil silica column with a mobile phase of acetonitrile-water containing trifluoroacetic acid. The MS/MS detection of the analytes was set in the positive ionization mode using electrospray ionization for sample introduction. The analyte and internal standard precursor-product ion combinations were monitored in the multiple-reaction monitoring mode. Assay calibration curves were linear in the concentration range 0.5--500 ng ml(-1) and 0.05--50 microg ml(-1) in plasma and urine, respectively. For the plasma assay, a 100 microl sample aliquot was subjected to extraction. To perform the urine assay, a 50 microl sample aliquot was used. The intra-day relative standard deviations at all concentration levels were <10%. The inter-day consistency was assessed by running quality control samples during each daily run. The limit of quantification was 0.5 ng ml(-1) in plasma and 0.05 microg ml(-1) in urine. The methods were utilized to support clinical pharmacokinetic studies in infants aged 0-12 months.     

Development of a routine method for the simultaneous confirmation and determination of clenbuterol in urine by minimal labeling isotope pattern deconvolution and GC-EI-MS

A novel and fast routine method for the simultaneous determination and confirmation of clenbuterol in bovine and human urine samples by gas chromatography electron ionization mass spectrometry (GC-EI-MS) has been developed. The method employs isotope dilution mass spectrometry (IDMS) and is based on a combination of minimal labeling (a single ¹³C label in the molecule) and isotope pattern deconvolution (IPD). This new methodology does not require the construction of a methodological calibration graph, and was compared with the classical IDMS procedure employed in clenbuterol analysis based on the use of a deuterated compound as internal standard (d₉-clenbuterol) and a calibration curve. The sample preparation consists of simple extraction with dichloromethane, which was dried and derivatized with chloro(chloromethyl)dimethylsilane, generating a cyclic dimethylsilamorpholine (DMS) derivative suitable for GC(EI)MS detection and identification. This compound produces five intense ions in the electron ionization source, which allow the presence of clenbuterol to be confirmed in just one analysis, as demanded by European Union directives. The accuracy of the method was studied by performing recovery experiments at different concentration levels (from 0.3 to 5 ng g⁻¹) in 5 mL bovine urine samples using two labeled compounds: an in-house-synthesized ¹³C₁-clenbuterol and a commercially available d₉-clenbuterol. The detection limit of the method in human urine was 0.050 ng g⁻¹ with a sample volume of 10 mL, and is thus suitable for antidoping control purposes. Finally, the ¹³C₁-clenbuterol standard was employed for the determination of clenbuterol in two reference materials, BCR-503 and BCR-504 (lyophilized bovine urine). The concentrations obtained were in agreement with the certified values, with a reproducibility of below 1% RSD.     

2-Methiopropamine, a thiophene analogue of methamphetamine: studies on its metabolism and detectability in the rat and human using GC-MS and LC-(HR)-MS techniques

2-Methiopropamine [1-(thiophen-2-yl)-2-methylaminopropane, 2-MPA], a thiophene analogue of methamphetamine, is available from online vendors selling “research chemicals.” The first samples were seized by the German police in 2011. As it is a recreational stimulant, its inclusion in routine drug screening protocols should be required. The aims of this study were to identify the phase I and II metabolites of 2-MPA in rat and human urine and to identify the human cytochrome-P450 (CYP) isoenzymes involved in its phase I metabolism. In addition, the detectability of 2-MPA in urine samples using the authors’ well-established gas chromatography–mass spectrometry (GC-MS) and liquid chromatography-linear ion trap-mass spectrometry (LC-MSⁿ) screening protocols was also evaluated. The metabolites were isolated from rat and human urine samples by solid-phase extraction without or following enzymatic cleavage of conjugates. The phase I metabolites, following acetylation, were separated and identified by GC-MS and/or liquid chromatography–high-resolution linear ion trap mass spectrometry (LC-HR-MSⁿ) and the phase II metabolites by LC-HR-MSⁿ. The following major metabolic pathways were proposed: N-demethylation, hydroxylation at the side chain and at the thiophene ring, and combination of these transformations followed by glucuronidation and/or sulfation. CYP1A2, CYP2C19, CYP2D6, and CYP3A4 were identified as the major phase I metabolizing enzymes. They were also involved in the N-demethylation of the analogue methamphetamine and CYP2C19, CYP2D6, and CYP3A4 in its ring hydroxylation. Following the administration of a typical user’s dose, 2-MPA and its metabolites were identified in rat urine using the authors’ GC-MS and the LC-MSⁿ screening approaches. Ingestion of 2-MPA could also be detected by both protocols in an authentic human urine sample.     

超高效液相色谱-三重四极杆质谱法测定血浆和尿液中马桑亭和马桑宁

建立了超高效液相色谱-三重四极杆质谱联用技术测定血浆和尿液中马桑中毒标志物马桑亭和马桑宁的方法。血浆和尿液样品经固相支持液液萃取法提取净化后，溶于15%（v/v）甲醇水溶液中，以Cortecs C₁₈色谱柱（100 mm×2.1 mm，1.6 μm）作为分析柱进行分离，电喷雾负离子多反应监测（MRM）模式下检测，以氟苯尼考作为内标物，基质工作曲线内标法定量。血浆和尿液中马桑亭和马桑宁的平均加标回收率为86.2%~110%，相对标准偏差为5.1%~14.6%（n=6），血浆中马桑亭和马桑宁的检出限（S/N=3）分别为0.01 μg/L和0.1 μg/L，尿液中马桑亭和马桑宁的检出限分别为0.03 μg/L和0.3 μg/L。本法简单、灵敏、准确，可用于血浆和尿液中马桑亭和马桑宁的中毒检测。     

Simultaneous determination of unmodified sevoflurane and of its metabolite hexafluoroisopropanol in urine by headspace sorptive extraction-thermal desorption-capillary gas chromatography-mass spectrometry

Unmodified sevoflurane and its metabolite, hexafluoroisopropanol (HFIP), have both been proposed as biomarkers of exposure in post-shift urine for operating room personnel exposed to inhalation anaesthetic sevoflurane. We used headspace sorptive extraction (HSSE) and thermal desorption-capillary GC-MS to assess sensitively both compounds in the urine matrix (after a HFIP deconjugation step). In GC-MS splitless mode, calibration plots (approximately 15-650 microg/L) were linear (r2 > 0.9910) and the limits of detection (1 microg/L for both biomarkers) showed increased sensitivity for HFIP with respect to the previously described headspace GC-MS method. The method was suitable for biological monitoring of both biomarkers of exposure to sevoflurane.