Human Biomonitoring of T-2 Toxin, T-2 Toxin-3-Glucoside and Their Metabolites in Urine through High-Resolution Mass Spectrometry.

The metabolic profile of T-2 toxin (T-2) and its modified form T-2-3-glucoside (T-2-3-Glc) remain unexplored in human samples. Therefore, the present study aimed to investigate the presence of T-2, T-2-3-Glc and their respective major metabolites in human urine samples (n = 300) collected in South Italy through an ultra-high performance liquid chromatography (UHPLC) coupled to Q-Orbitrap-HRMS methodology. T-2 was quantified in 21% of samples at a mean concentration of 1.34 ng/mg Crea (range: 0.22-6.54 ng/mg Crea). Almost all the major T-2 metabolites previously characterized in vitro were tentatively found, remarking the occurrence of 3'-OH-T-2 (99.7%), T-2 triol (56%) and HT-2 (30%). Regarding T-2-3-Glc, a low prevalence of the parent mycotoxin (1%) and its metabolites were observed, with HT-2-3-Glc (17%) being the most prevalent compound, although hydroxylated products were also detected. Attending to the large number of testing positive for T-2 or its metabolites, this study found a frequent exposure in Italian population.

Multimycotoxin Exposure Assessment in UK Children Using Urinary Biomarkers-A Pilot Survey.

Cereal foods are commonly contaminated with multiple mycotoxins resulting in frequent human mycotoxin exposure. Children are at risk of high-level exposure because of their high cereal intake relative to body weight. Hence, this study aims to assess multimycotoxin exposure in UK children using urinary biomarkers. Spot urines (n = 21) were analyzed for multimycotoxins (deoxynivalenol, DON; nivalenol, NIV; ochratoxin A, OTA; zearalenone, ZEN; α-zearalenol, α-ZEL; ß-zearalenol, ß-ZEL; T-2 toxin, T-2; HT-2 toxin, HT-2; and aflatoxin B1 and M1, AFB1, AFM1) using liquid chromatography-coupled tandem mass spectrometry. Urine samples frequently contained DON (13.10 ± 12.69 ng/mL), NIV (0.36 ± 0.16 ng/mL), OTA (0.05 ± 0.02 ng/mL), and ZEN (0.09 ± 0.07 ng/mL). Some samples (1-3) contained T-2, HT-2, α-ZEL, and ß-ZEL but not aflatoxins. Dietary mycotoxin estimation showed that children were frequently exposed to levels exceeding the tolerable daily intake (52 and 95% of cases for DON and OTA). This demonstrates that UK children are exposed to multiple mycotoxins through their habitual diet.

Prevalence of Selenium, T-2 Toxin, and Deoxynivalenol in Kashin-Beck Disease Areas in Qinghai Province, Northwest China.

The aim of this study was to determine the levels of selenium, T-2 toxin, and deoxynivalenol (DON) contamination in Kashin-Beck disease (KBD) areas and provide information for understanding the high prevalence of KBD in Qinghai Province. A total of 183 subjects were chosen in a KBD-prevalent county (Guide County) and a non-KBD county (Huangzhong County) in Qinghai Province, northwestern China, and the samples of wheat flour, soil, drinking water and blood, urine, and hair of children were collected from these residents. The selenium concentrations from all these sources were determined using atomic fluorescence spectrophotometry. The levels of T-2 toxin and DON contamination in the wheat flour samples were assayed using HPLC-MS/MS. The average selenium content in the soil, drinking water, and wheat flour samples from KBD areas were 26.93 ± 10.06 µg/kg, 0.097 ± 0.038 µg/L, and 9.50 ± 7.17 µg/kg, respectively. Among these, the selenium levels in the drinking water and wheat flour samples from the KBD endemic county were significantly lower than those from the non-KBD county. For the selenium nutrient status, only the hair selenium concentration of children from the KBD endemic county was significantly lower than that from the non-KBD county. The contents of T-2 toxin in all wheat samples were below the detection limit (0.4 µg/kg). The levels of DON contamination in wheat flour samples from KBD and non-KBD children's households within the KBD endemic county were relatively higher, with average levels of 302 ± 49 and 280 ± 48 µg/kg, respectively. The DON level of wheat flour samples from the children's households in the non-KBD county was significantly lower than that from the KBD endemic county. These results suggest that the lower selenium status in Guide County still remains. While the selenium nutritional status of the local children has improved to some extent, partly due to the introduction of food produce from nonlocal areas. DON contamination in the wheat flour may be involved in the fluctuating high prevalence rates of KBD in children in the KBD endemic Guide County in Qinghai Province.

Determination of mycotoxin exposure in Germany using an LC-MS/MS multibiomarker approach.

SCOPE: In this study, the exposure of a German population (n = 101) to mycotoxins was assessed using an LC-MS/MS urinary multibiomarker approach. Food consumption of the participants was documented with a food frequency questionnaire to correlate mycotoxin exposure with individual nutritional habits. METHODS AND RESULTS: The presence of 23 urinary biomarkers including trichothecenes (deoxynivalenol (DON), DON-3-glucuronide (DON-3-GlcA), T-2 toxin, HT-2 toxin (HT-2, HT-2-toxin-4-glucuronide (HT-2-GlcA), fumonisins (fumonisin B1, fumonisin B2), aflatoxins (aflatoxin B1, aflatoxin G2, aflatoxin B2, aflatoxin M1), zearalenone and derivatives (zearalanone, α-zearalenol, ß-zearalenol, zearalenone-14-O-glucuronide, zearalanone-14-O-glucuronide, α-zearalenol-14-O-glucuronide/ß-zearalenol-14-O-glucuronide), ochratoxin A, ochratoxin alpha, enniatin B and dihydrocitrinone was evaluated using a validated, sensitive "dilute and shoot"-LC-MS/MS method applying Scheduled MRM(TM) technology. Six mycotoxins and urinary metabolites were detected (DON, DON-3-GlcA, zearalenone-14-O-glucuronide, T-2 toxin, enniatin B, and dihydrocitrinone) in 87% of the samples in single- or co-occurence. Only DON and DON-3-GlcA were detectable in quantifiable amounts. A provisional mean daily intake of 0.52 µg DON/kg body weight was calculated. No statistical evidence for the correlation of staple food intake and urinary biomarker concentration could be determined. CONCLUSION: The results of this study suggest a low everyday exposure of the investigated German population to mycotoxins, but reveal peak exposures above the widely accepted tolerable daily intake to DON in parts of the population.

Exposure assessment approach through mycotoxin/creatinine ratio evaluation in urine by GC-MS/MS.

In this pilot survey human urine samples were analyzed for presence of 15 mycotoxins and some of their metabolites using a novel urinary multi-mycotoxin GC-MS/MS method following salting-out liquid-liquid extraction. Fifty-four urine samples from children and adults residents in Valencia were analyzed for presence of urinary mycotoxin and expressed in gram of creatinine. Three out of 15 mycotoxins were detected namely, HT-2 toxin, nivalenol and deoxynivalenol (DON). 37 samples showed quantifiable values of mycotoxins. Co-occurrence of these contaminants was also observed in 20.4% of assayed samples. DON was the most frequently detected mycotoxin (68.5%) with mean levels of 23.3 µg/g creatinine (range: 2.8-69.1 µg/g creatinine). The levels of urinary DON were used to carry out an exposure assessment approach. 8.1% of total subjects were estimated to exceed the DON provisional maximum tolerable daily intake (PMTDI) (1 µg/kg b.w.). Two out of 9 exposed children exceeded the DON PMTDI thus, making them the most exposed based on the urinary results.

Metabolic pathways of T-2 toxin in in vivo and in vitro systems of Wistar rats.

In the present study, metabolites of T-2 toxin in in vivo and in vitro systems of Wistar rats were identified and elucidated by ultraperformance liquid chromatography-quadrupole/time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS). Expected and unexpected metabolites were detected by Metabolynx(XS) software, which could automatically compare MS(E) data from the sample and control. A total of 19 metabolites of T-2 toxin were identified in this research, 9 of them being novel, which were 15-deacetyl-T-2, 3'-OH-15-deacetyl-T-2, 3',7-dihydroxy-T-2, isomer of 3',7-dihydroxy-T-2, 7-OH-HT-2, isomer of 7-OH-HT-2, de-epoxy-3',7-dihydroxy-HT-2, 9-OH-T-2, and 3',9-dihydroxy-T-2. The results showed that the main metabolic pathways of T-2 toxin were hydrolysis, hydroxylation, and de-epoxidation. In addition, the results also revealed one novel metabolic pathway of T-2 toxin, hydroxylation at C-9 position, which was demonstrated by the metabolites 9-OH-T-2 and 3',9-dihydroxy-T-2. In addition, hydroxylation at C-9 of T-2 toxin was also generated in in vitro of liver systems. Interestingly, several metabolites of hydroxylation at C-7 of T-2 toxin were also detected in in vivo male Wistar rats, but they were not found in in vivo female rats and in in vitro systems of Wistar rats.

Development and application of salting-out assisted liquid/liquid extraction for multi-mycotoxin biomarkers analysis in pig urine with high performance liquid chromatography/tandem mass spectrometry.

Direct determination of urinary mycotoxins is a better approach to assess individual's exposure than the indirect estimation from average dietary intakes. In this study, a new analytical method was developed and validated for simultaneous analysis of aflatoxin B1, deoxynivalenol, fumonisin B1, ochratoxin A, zearalenone and T2 toxin and their metabolites in pig urine. In total 12 analytes were selected. A salting-out assisted liquid-liquid extraction procedure was used for sample preparation. High performance liquid chromatography/tandem mass spectrometry was used for the separation and detection of all the analytes. The extraction recoveries were in a range of 70-108%, with the intra-day relative standard deviation and inter-day relative standard deviation lower than 25% for most of the compounds at 3 different concentration levels. Meanwhile the method bias for all the analytes did not exceed 20%. The limits of quantification ranged from 0.07ngmL(-1) for ochratoxin A to 3.3ngmL(-1) for deoxynivalenol. Matrix effect was evaluated in this study and matrix-matched calibration was used for quantification. The developed method was also validated for human urine as an extension of its application. Finally, the developed method was applied in a pilot study to analyze 28 pig urine samples. Deoxynivalenol, aflatoxin B1, fumonisin B1 and ochratoxin A were detected in these samples.

A high-performance liquid chromatographic method for determining [3H]T-2 and its metabolites in biological fluids of the cynomolgus monkey.

High-performance liquid chromatography (HPLC) was used to separate, identify, and quantitate the trichothecin mycotoxin, T-2 (4 beta,15-diacetoxy-3 aopha-hydroxy-8 alpha [3-methyl-butyryloxy]-12,13-epoxy delta 9-trichothecin), and its metabolites in plasma and urine samples from cynomolgus monkeys treated with the toxin. A 15-min gradient elution system was developed to separate and measure radiolabeled T-2 mycotoxin and its metabolites. The HPLC technique for separating T-2 and its metabolites was compared with thin-layer chromatography. Samples from the in vitro metabolism of T-2 by plasma and urine were included as controls and as a measure of the toxin's stability in biological samples. Within 5 min, 22% of the plasma radiolabeled T-2 toxin was detected as metabolites after an intravenous administration of [3H] T-2 toxin to cynomolgus monkeys. By 24 h post-exposure, there was no parent T-2 toxin detected in plasma or urine. T-2 tetraol was the major metabolite detected in the plasma and urine of monkeys. Other metabolites observed in urine up to 5 days after exposure were 3'OH-T-2 and 3'OH-HT-2. We conclude that T-2 toxin was rapidly metabolized to more polar metabolites, which were eliminated in urine.

Use of deuterated internal standards for quantitation of T-2 and HT-2 toxins in human blood by tandem mass spectrometry.

Deuterated acetyl derivatives (3-trideutero-acetyl-T-2 and 15-trideutero-HT-2) were prepared for use as internal standards for the quantitation of T-2 and HT-2 in blood by tandem mass spectrometry. The method used was multiple reaction monitoring (MRM), which essentially involves the selection of a parent ion for analysis followed by monitoring of the daughter ions generated by collision activated decomposition. The parent ions chosen for the trifluoroacetate derivative of T-2 and HT-2 were m/z+ 478 and 532, respectively. Both parents yield the same daughter ions, i.e., 180, 138, and 121. HT-2 and T-2 were added to blood extracts in amounts ranging from 1 to 20 ppb. The limit of detection is about 0.5 ppb with an effective detection limit of 1.0 ppb in a range of 1-20 ppb. The recovery is about 90%. This method can be used by veterinarians for purposes of diagnostics. It can be used for urine as well as blood.

Analysis of T-2 toxin in a biological matrix using multiple reaction monitoring.

The traditional analysis of a biological mixture by mass spectrometry involves the union of a gas liquid chromatograph with a mass spectrometer and analysis of the resolved effluent by either full scan or the recording of selected ions. The latter methodology is sensitive and selective but suffers from the interference presented by the biological matrix. With the advent of tandem mass spectrometry, greater flexibility in the elimination of the effects of a biological matrix is possible. The example used here is that of the trifluoroacetate derivative of T-2 toxin. Thus, a single or multiple selection of parent ions (m/z+ 478) is made and allowed to pass through the first analyzer (sectoring portion) of the tandem mass spectrometer into the second mass spectrometer, in this case a quadrupole. Here the parent fragment undergoes collision-activated decomposition, under the influence of argon gas and voltage (collision energy) into daughter ions which are detected by the third mass spectrometer (quadrupole). The daughters generated from the parent m/z+ 478 of T-2-TFA are 180, 138 and 121. They are unique and give definitive proof for the presence of T-2 toxin. There is the possibility that other 478 fragments may be present in the mixture that have the same retention time as T-2-TFA toxin. In this case, the daughters generated will be different from that of T-2. The method is called multiple reaction monitoring. It is highly accurate and can detect T-2-TFA in blood or urine at a concentration of 1 ppb.

Enzyme-linked immunosorbent assay for T-2 toxin metabolites in urine.

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of total T-2 toxin metabolites in urine was developed. The assay involves coating anti-3-acetyl-neosolaniol-hemisuccinate-bovine serum albumin conjugate (anti-3-Ac-NEOS-HS-BSA) antibody to the ELISA plate and using 3-Ac-NEOS-HS-peroxidase as the enzyme marker. Competitive ELISA revealed that the antibody had good cross-reactivity with acetyldiacetoxyscirpenol (Ac-DAS), T-2 tetraol tetraacetate, 3'-OH-Ac-T-2, 3-Ac-NEOS, and 3,4,15-triacetyl-12,13-epoxytrichothec-9-en-8-one (Ac-T-2-8-one), but less cross-reactivity with Ac-T-2 toxin and T-2 toxin. All metabolites of T-2 toxin in urine were converted to T-2 tetraol tetraacetate (T-2-4ol-4Ac) by acetylation of the sample extract before ELISA. To test the ELISA accuracy, a radioimmunoassay (RIA) was performed simultaneously. The linear portion of the standard curve of this direct ELISA for T-2-4ol-4Ac was 0.2-2.0 ng/mL, which was 10 times more sensitive than RIA. The minimum detection level for T-2-4ol-4Ac was 0.02 ng/mL (0.4 pg/assay) in the absence of urine sample. The overall analytical recoveries for T-2 toxin, HT-2, T-2-4ol, 3'-OH-HT-2, NEOS, and a mixture of these 5 toxins added to the urine samples in the ELISA at concentrations of 0.05 and 0.2 ng/mL were 87 and 94%, respectively.

Stability of T-2, HT-2, and T-2 tetraol in biological fluids.

The stabilities of tritium-labeled T-2, HT-2, and T-2 tetraol were studied in blood and urine at -70 degrees, 4 degrees, and 23 degrees C for 6 months in the presence of EDTA or NaF. Samples were counted with a radiochromatographic scanner and results indicated the stability of T-2 tetraol greater than T-2 greater than HT-2. Toxins were most stable when stored at -70 degrees C, in the presence of NaF, and in urine (pH 6). They were less stable in saline (control, pH 7) and least stable in blood (pH 8). These results suggest that urine and T-2 tetraol are the biological fluid and metabolite of choice for diagnostic purposes.

Pharmacokinetics of T-2 tetraol, a urinary metabolite of the trichothecene mycotoxin, T-2 toxin, in dog.

1. The urinary metabolites of T-2 toxin were identified and analysed quantitatively after i.v. administration to dogs. 2. A new routine assay for T-2 tetraol was developed and a pharmacokinetic study was carried out on this final hydrolytic metabolite of T-2 toxin. T-2 tetraol was excreted in urine for 2-3 days. Its 'sigma minus' plot demonstrated a significantly longer apparent half-life than its precursors (T-2 toxin and HT-2 toxin). This fact was explained by extraplasma binding causing prolongation of the metabolism and excretion of T-2 toxin metabolites. 3. The urinary metabolites of T-2 toxin were: HT-2 toxin, T-2 triol and T-2 tetraol. The metabolites were excreted in free and conjugated forms. In two dogs T-2 toxin was found in the urine in an amount which accounts for 3.2 and 16% of the administered dose respectively. The cumulative amount of the identified metabolites and toxins formed in the urine ranged from 9.7 to 17.3% in four dogs and 44.7% in one dog.

Simultaneous analysis of T-2 toxin and HT-2 toxin by an indirect enzyme-linked immunosorbent assay.

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of HT-2 toxin in the presence or absence of T-2 toxin is described. In the indirect ELISA, the relative cross-reactivities of antibodies against T-2 toxin (anti-T-2) with T-2 toxin and HT-2 toxin were 1 and 0.1, whereas anti-HT-2 cross-reactivities with T-2 toxin and HT-2 toxin were 0.33 and 1, respectively. Using such relationships, a formula was established that could be used to calculate the individual toxin concentration in a mixed sample after experimentally analyzing for T-2 and HT-2 toxins in the 2 indirect ELISAs. This method was tested by analyzing urine samples spiked with HT-2 toxin alone and samples spiked with both T-2 toxin and HT-2 toxin. A cleanup protocol for treatment of urine samples before ELISA was also established. The overall analytical recovery of HT-2 toxin when it was added at concentrations of 0.1-10 parts per billion (ppb) to the urine samples was ca 89%. When both T-2 and HT-2 toxins were added to the urine samples at equal concentrations of 0.5 to 5.0 ppb, their recoveries were 112 and 109%, respectively.

Analysis of some metabolites of T-2 toxin, diacetoxyscirpenol and deoxynivalenol by thermospray high-performance liquid chromatography-mass spectrometry.

Detection and identification of mycotoxin metabolites is a very challenging task. In order to achieve adequate sensitivity and specificity an analytical technique must overcome serious matrix interferences. Gas chromatography-mass spectrometry (GC-MS) which has the sensitivity and specificity to detect and identify mycotoxin metabolites requires hydrolysis of conjugated metabolites as well as derivatization. Thermospray high-performance liquid chromatography-mass spectrometry (HPLC-MS) offers the sensitivity, specificity, and structural information to detect and identify some mycotoxin metabolites in fecal and urine samples without derivatization. The mycotoxins evaluated in this study include deoxynivalenol (DON), T-2 toxin, and diacetoxyscirpenol. The de-epoxy and hydroxy metabolites of each toxin and the glucuronide conjugate of DON were isolated, extracted, and analyzed to detect their occurrence in animals. The thermospray mass spectra of the toxins showed an [M + H]+ ion and numerous structurally significant fragment ions in the positive ion detection mode. Negative ion detection exhibited primarily [M + acetate]- cluster ions with less fragmentation than observed by positive ion detection. The operation of the interface in the filament-on mode greatly increased the sensitivity in both positive and negative ion detection mode. Detection limits of 50-500 pg injected on column are obtained for these toxins and their metabolites using multiple ion detection. The urine and fecal extracts from rats, hens, and cows did not interfere with the HPLC-MS analysis for the specific metabolites or the glucuronide conjugate.

Comparison of in vivo and in vitro percutaneous absorption of T-2 toxin in guinea pigs.

The fate and distribution of T-2 were examined in 6 guinea pigs. T-2 (1.2 micrograms/cm2), in methanol or DMSO, was painted onto the shaved backs of guinea pigs, a screen barrier was applied, urine and feces were collected daily and the guinea pigs were killed after 48 hr. Disks of skin (lateral to the in vivo site of application) were excised from the guinea pigs and used for in vitro penetration studies with static diffusion cells. Skin excised from 6 additional guinea pigs was used for penetration studies with flow-through diffusion cells. For in vitro studies, T-2 dissolved in methanol or DMSO was applied to the epidermal surfaces and the appearance of penetrant in receptor fluid bathing the dermal surfaces was monitored for 48 hr. Metabolism of T-2 was measured by using thin layer radiochromatography to identify metabolites. In the in vivo study, mean cutaneous absorption (n = 3) after 48 hr (expressed as per cent dose) was 22.5 and 51.9 for the methanol and DMSO groups, respectively. In vitro cutaneous penetration for static diffusion cells was 3.9 and 38.4 for the methanol and DMSO groups. For flow-through diffusion cells, mean penetration (n = 9) was 14.6 and 42.6 for the methanol and DMSO groups. Urinary metabolites of T-2 were T-2 triol, 3' OH-HT-2, T-2 tetraol, the glucuronide conjugate of HT-2 and several more polar metabolites. The main metabolite of T-2 in the receptor fluid bathing the dermal surfaces of excised skin was HT-2.

Detection of trace levels of trichothecene mycotoxins in human urine by gas chromatography-mass spectrometry.

A method is described for the simultaneous detection of the trichothecene mycotoxins T-2, HT-2, T-2 tetraol, diacetoxyscirpenol, 15-monoacetoxyscirpendiol, scirpentriol, nivalenol and deoxynivalenol, in human urine. Samples were extracted from Clin Elut columns and cleaned up using reversed-phase Sep-Pak C18 cartridges. Trichothecenes were derivatised as their heptafluorobutyryl esters, and detected by gas chromatography-mass spectrometry-selected-ion monitoring using electron impact ionisation. The method was validated by the analysis of 22 urine samples, spiked and submitted "blind" for analysis by another laboratory. An alternative gas chromatography-mass spectrometry method using negative ion chemical ionisation is also described and a preliminary comparison of the two methods made. The methods enabled levels down to 1 ppb to be detected, with confirmation of identity at levels between 2 and 5 ppb, depending on the toxin.

Rapid screening procedure for the detection of trichothecenes in plasma and urine.

A rapid and easy procedure to screen for trichothecenes in plasma and urine is presented. The toxins are extracted using a Clin-Elut column, hydrolyzed to their corresponding parent alcohols and cleaned up with a silica cartridge followed by derivatization for gas chromatographic analysis. The detection of any of the parent alcohols in plasma or urine would indicate an exposure to trichothecenes. Recoveries in urine are between 78 and 119% at levels of 50-1000 ng/ml and recoveries in plasma are between 80 and 116% at levels of 50-500 ng/ml. The limit of detection is better than 25 ppb.

Identification of iso-TC-1 as a new T-2 toxin metabolite in cow urine.

The structure of a new metabolite T-2 toxin (iso-TC-1) has been established as 3,15-diacetoxy-4-hydroxy-8(3-methyl-3'-hydroxy-butyryloxy)-12, 13-epoxytrichothec-9-ene. The compound is an isomer of TC-1 (a recently isolated T-2 derivative) in which the hydroxy and acetoxy groups at the C-3 and C-4 positions, respectively, are reversed. Direct probe analysis by electron impact (EI) of the underivatized iso-TC-1, as well as EI, positive chemical ionization (CI) in methane, and positive CI in ammonia of its trimethylsilylether or trifluoroacetate provided evidence to support the structure assignment of the new metabolite. The mass spectra of iso-TC-1 were compared with those of TC-1, T-2 toxin and iso-T-2 toxin (the isomer of T-2 toxin having reversed substituents at C-3 and C-4) with regard to molecular weight and fragments involving the substituents at C-3, C-4, C-8 and C-15. Although the two isomers, TC-1 and iso-TC-1, were not easily resolved by thin layer chromatography (TLC), a very good separation of their trimethylsilyl and trifluoroacetate derivatives was obtained by capillary gas chromatography. Acetylation of TC-1 or iso-TC-1 gave the same product. Iso-TC-1 is one of the main products of T-2 metabolism in the cow (more abundant than TC-1) and is found in the urine.

Radioimmunoassay of T-2 toxin in biological fluids.

A simple radioimmunoassay (RIA) for detection of T-2 toxin in biological fluids was developed. The method, which involves a Sep-Pak C18 reverse phase column cleanup step before RIA, can detect 0.5 ppb T-2 toxin in serum and 2.5 ppb T-2 toxin in urine and milk. Recoveries for toxin added to serum and urine or milk samples were 87-106% in the 0.5-20 ppb range and 88-117% in the 2.5-20ppb range, respectively. If samples were not cleaned up, false positive results prevented positive detection at concentration less than 2.5 ppb in urine and milk.