Determination of eprosartan in human plasma and urine by LC/MS/MS

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of eprosartan in human plasma and urine. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on a CAPCELL PAK C18 column (50 mmx2.0 mm, 5 microm, Shiseido). A mobile phase was consisted of 0.5% formic acid in water and 0.5% formic acid in acetonitrile (72:28). Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 5 to 2000 ng/mL in human plasma and from 0.25 to 50 microg/mL in urine, were fitted to a 1/x weighted quadratic regression model. The method proved to be accurate, specific and sensitive enough to be successfully applied to a pharmacokinetic study.     

A sensitive assay for simultaneous determination of plasma concentrations of valganciclovir and its active metabolite ganciclovir by LC/MS/MS

A protein precipitation, liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of valganciclovir and its active metabolite ganciclovir in human plasma. The solvent system also served as a protein precipitation reagent. The chromatographic separation was achieved on an Aquasil C18 column (50 mm x 2.1mm, 5 microm). A linear gradient mobile phase between 0.02% formic acid and methanol was used. Detection was by positive ion electrospray tandem mass spectrometry on a Sciex API3000. The standard curves, which ranged from 4 to 512 ng/mL for valganciclovir and from 0.1 to 12.8 microg/mL for ganciclovir, were fitted to a 1/x weighted quadratic regression model. The method was proved to be accurate, specific and sensitive enough and was successfully applied to a pharmacokinetic study.     

Determination of the HIV integrase inhibitor, MK-0518 (raltegravir), in human plasma using 96-well liquid-liquid extraction and HPLC-MS/MS

An HPLC-MS/MS method was developed for the determination of MK-0518 (raltegravir), an HIV integrase inhibitor, in human plasma over the concentration range of 2-1000 ng/mL. Stable isotope labeled (13)C(6)-MK-0518 was used as an internal standard. The sample preparation procedure utilized liquid-liquid extraction with hexane:methylene chloride in the 96-well format with a 200 microL plasma sample size. The compounds were chromatographed on an Ace C(18) (50 x 3.0 mm, 3 microm, titanium frits) column with 42.5/57.5 (v/v %) 0.1mM EDTA in 0.1% formic acid/methanol mobile phase at a flow rate of 0.5 mL/min. Multiple reaction monitoring of the precursor-to-product ion pairs for MK-0518 (m/z 445-->109) and (13)C(6)-MK-0518 (m/z 451-->367) on an Applied Biosystem API 4000 HPLC-MS/MS was used for quantitation. Intraday precision of standard curve concentrations in five different lots of control plasma was within 3.2%, while accuracy ranged from 94.8 to 106.8%. The mean extraction recovery of spiked plasma samples was 87%. Quality control (QC) samples were stored at -20 degrees C. Initial within day analysis showed QC accuracy within 7.5% of nominal with precision of 3.1% or less. The plasma QC samples were demonstrated to be stable for up to 23 months at -20 degrees C. The method described has been used to support over 18 clinical studies during Phase I through III of clinical development.     

LC-MS/MS determination in rabbit plasma of the main photoproduct of RLP068/Cl, a cationic sensitizer proposed for photodynamic therapy (PDT) of microbial infections

The clinical development of a sensitizer for photodynamic therapy (PDT) requires the structural identification of the photoproducts and their quantification in biological fluids and tissues. We describe the LC-MS identification of the most important photoproducts of a cationic phthalocyanine sensitizer (RLP068/Cl) and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of the main photoproduct (the cationic phthalimide derivative 3-[(1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl)oxy]-N,N,N-trimethylbenzenaminium chloride) in rabbit plasma. The tri-deuterated product was used as co-eluting internal standard. The cationic photoproduct was isolated from plasma samples by protein precipitation with perchloric acid in methanol (7%, v/v). HPLC step was performed on a Phenomenex Synergi Hydro-RP column (20 mm x 2.0 mm, 2 microm particles) with a mobile phase of 0.5% (v/v) aqueous TFA/methanol (85:15, v/v). Flow rate was 0.2 mL/min and 40 microL injection were performed. Run time was 10 min. Detection was achieved by means of a Bruker Esquire 3000+ ion trap mass spectrometer equipped with an ESI source working in positive mode. A multiple reaction monitoring method following the transitions 297.1 --> 282.1 for the analyte and 300.1 --> 282.1+285.1 for the internal standard was used. The analytical method was validated over the concentration range 0.46-91.2 ng/mL and lower limits of detection (LLOD) and quantification (LLOQ) respectively of 0.2 and 0.5 ng/mL were found.     

A rapid and sensitive method for determination of sorafenib in human plasma using a liquid chromatography/tandem mass spectrometry assay

A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sorafenib in human plasma. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma with 0.5 mL acetonitrile. Analysis of the compounds of interest including the internal standard ([(2)H(3)(15)N] sorafenib) was achieved on a Waters X-Terra C(18) (150 mm x 2.1mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile/10 mM ammonium acetate (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.2 mL/min for 6 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 7.3-7260 ng/mL for the human plasma samples with values for the coefficient of determination of >0.96. The values for both within day and between day precision and accuracy were well within the generally accepted criteria for analytical methods (<15%).     

Quantification of cyclizine and norcyclizine in human plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

A rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for quantification of cyclizine and its main metabolite norcyclizine in human plasma. Samples were prepared by protein precipitation with acetonitrile and cinnarizine was used as internal standard (recovery >87%). The analytes were eluted from a C8 50 mm×2.0 mm analytical column using a linear gradient of methanol and 0.05% formic acid with a total analysis time of 4 min. Analytes were detected by MS/MS using electrospray ionisation in the positive mode with multiple reactions monitoring (MRM) of the precursor ion/product ion transitions 267.2/167.2 for cyclizine and 253.2/167.2 for norcyclizine. Matrix effects were negligible. Standard curves for cyclizine and norcyclizine were linear (r(2)≥0.996) over the range 2-200 ng/mL, with 2 ng/mL representing the lower limit of quantification. Relative standard deviations were <14% for intra- and inter-day precision and the accuracy was within ±8%. The assay was successfully applied to a clinical study.     

A rapid and sensitive HPLC-MS/MS analysis and preliminary pharmacokinetic characterization of sibiricaxanthone F in rats

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantifying sibiricaxanthone F (SF) in rat plasma following oral and intravenous dosings. After addition of the internal standard (IS) kaempferol and the antioxidant, 20% ascorbic acid, plasma samples were precipitated with acetonitrile and separated on an Aglient Zorbax XDB-C(18) column (50 mm × 4.6mm I.D., 2.1 μm) with gradient acetonitrile and water (both containing 0.01% formic acid) as the mobile phase. The detection was performed on a Sciex API 4000 LC-MS/MS with electrospray ionization (ESI) inlet in the negative multiple reaction monitoring (MRM) mode. Good linearity was achieved over the concentration range of 0.5-500.0ng/mL (r>0.996). Intra- and inter-day precisions were less than 7.60%, and accuracy ranged from 97.18% to 99.84%. The lower limit of quantification for SF was 0.5 ng/mL, and analytes were stable under various conditions (during freeze-thaw, at room temperature and under deep-freeze conditions). This validated method was successfully applied to the preliminary pharmacokinetic study of SF in rats for the first time, and the absolute bioavailability of SF was found to be only 0.22 ± 0.15%.     

Simultaneous determination of codeine, ephedrine, guaiphenesin and chlorpheniramine in beagle dog plasma using high performance liquid chromatography coupled with tandem mass spectrometric detection: application to a bioequivalence study

A rapid and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of codeine, ephedrine, guaiphenesin and chlorpheniramine in beagle dog plasma has been developed and validated. Following liquid-liquid extraction, the analytes were separated on a reversed-phase C(18) column (150 mm × 2.0 mm, 3 μm) using formic acid:10 mM ammonium acetate:methanol (0.2:62:38, v/v/v) as mobile phase at a flow rate of 0.2 mL/min and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode. The method was linear for all analytes over the following concentration (ng/mL) ranges: codeine 0.08-16; ephedrine 0.8-160; guaiphenesin 80-16,000; chlorpheniramine 0.2-40. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. It is the first time that the validated HPLC-MS/MS method was successfully applied to a bioequivalence study in 6 healthy beagle dogs.     

Quantitation of tetramethylene disulfotetramine in human urine using isotope dilution gas chromatography mass spectrometry (GC/MS and GC/MS/MS)

Tetramethylene disulfotetramine (tetramine) is a rodenticide associated with numerous poisonings was extracted and quantified in human urine using both gas chromatography/mass spectrometry (GC/MS) and GC/tandem mass spectrometry (MS/MS). 1200 μL samples were prepared using a ¹³C₄-labeled internal standard, a 96-well format, and a polydivinyl-benzene solid phase extraction sorbent bed. Relative extraction recovery was greater than 80% at 100 ng/mL. Following extraction, samples were preconcentrated by evaporation at 60 °C, and reconstituted in 50 μL acetonitrile. One-microliter was injected in a splitless mode on both instruments similarly equipped with 30 m × 0.25 mm × 25 μm, 5% phenyl-methylpolysiloxane gas chromatography columns. A quantification ion and a confirmation ion (GC/MS) or analogous selected reaction monitoring transitions (GC/MS/MS) were integrated for all reported results. The method was characterized for precision (5.92–13.4%) and accuracy (96.4–111%) using tetramine-enriched human urine pools between 5 and 250 ng/mL. The method limit of detection was calculated to be 2.34 and 3.87 ng/mL for GC/MS and GC/MS/MS, respectively. A reference range of 100 unexposed human urine samples was analyzed for potential endogenous interferences on both instruments—none were detected. Based on previous literature values for tetramine poisonings, this urinary method should be suitable for measuring low, moderate, and severe tetramine exposures.     

A rapid, sensitive and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry method for determination of fenretinide (4-HPR) in plasma

A simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method using an atmospheric pressure chemical ionization source (APCI) for the quantification of fenretinide (4-HPR) in mouse plasma was developed and validated. After a simple protein precipitation of plasma sample by acetonitrile, 4-HPR was analyzed by LC-APCI-MS/MS. High-performance liquid chromatography (HPLC) separation was conducted on a Hypurity C18 column (50mmx2.1mm, 5microm) with a flow rate 0.60mL/min using a gradient mobile phase comprised of 0.05% formic acid in water (A) and methanol (B), and a run time of 4.5min. The elimination of a tedious sample preparation process and a shorter run time substantially reduced total analysis time. The method was linear over the range 0.5-100ng/mL, with r>0.998. The intra- and inter-assay precisions were 1.4-9.2% and 5.1-8.2%, respectively, and the intra- and inter-assay accuracies were 93.9-98.6% and 92.7-95.3%, respectively. The absolute recoveries were 90.3% (1.5ng/mL), 97.0% (7.5ng/mL) and 92.1% (75.0ng/mL) for 4-HPR, and 99.1% for the internal standard (150ng/mL). The analytical method had excellent sensitivity using a small sample volume (30microL) with the lower limit of quantification (LLOQ) 0.5ng/mL. This method is robust and has been successfully employed in a pharmacokinetic study of 4-HPR in a mouse xenograft model of neuroblastoma.     

Anion exchange SPE and liquid chromatography-tandem mass spectrometry in GHB analysis

In this study, the extraction of γ-hydroxybutyrate (GHB) from urine using solid-phase extraction (SPE) is described. SPE was performed on anion exchange columns after samples of urine had been diluted with de-ionized water. After application of the diluted samples containing GHB-d(6) as an internal standard, the sorbent was washed with deionized water and methanol and dried. The GHB was eluted from the SPE column with a solvent consisting of methanol containing 6% glacial acetic acid. The eluent was collected, evaporated to dryness, and dissolved in mobile phase (100 μL) for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in negative multiple reaction monitoring (MRM) mode. Liquid chromatography was performed in gradient mode employing a biphenyl column and a mobile phase consisting of acetontitrile (containing 0.1% formic acid) and 0.1% aqueous formic acid. The total run time for each analysis was less than 5 min. The limits of detection/quantification for this method were determined to be 50 and 100 ng/mL, respectively. The method was found to be linear from 500 ng/mL to 10,000 ng/mL (r(2)>0.995). The recovery of GHB was found to be greater than 75%. In this report, results of authentic urine samples analyzed for GHB by this method are presented. GHB concentrations in these samples were found to be range from less than 500 ng/mL to 5110 ng/mL.     

Determination of iohexol clearance by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)

We have developed a simple, rapid, and accurate HPLC-MS/MS method for the determination of iohexol in serum. The column used was a Zorbax Eclipse XDB-C8 (100 mm x 2.1 mm i.d., 3.5 microm). Mobile phases consisted of water containing 2mM ammonium acetate and 0.1% formic acid (A) and methanol containing 2 mM ammonium acetate and 0.1% formic acid (B). After simple protein precipitation with ZnSO4, serum samples were mixed with I.S. (bromperidol) and centrifuged for 3 min. The obtained extraction recovery at three levels was 94.6-107.4%. Quantitative analysis was performed in the multiple reaction-monitoring mode (m/z 822.0-->804.0 for iohexol, 420.1-->122.7 for I.S.) with the total running time of 3 min for each sample. The assay was linear between 0.5 and 1500 microg/mL (r2 > 0.997). The intra- and inter-assay coefficient of variations were 2.4-6.2% and 5.5-6.5%, respectively. Our method provided sufficient analytical range and specificity for the 210 clinical samples analyzed.     

Quantification of sunitinib in human plasma by high-performance liquid chromatography-tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sunitinib in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.2mL of plasma with 4.0mL tert-butyl-methyl-ether extraction solution containing 25ng/mL of the internal standard clozapine. Separation of compounds was achieved on a C18 (50mmx2.1mm i.d., 3.5microm) analytical column using a mobile phase consisting of acetonitrile/H20 (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.150mL/min for 3min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves in human plasma were generated over the range of 0.2-500ng/mL with values for the coefficient of determination of >0.9950. Within- and between day precision and accuracy were < or =10%. The method was applied to the quantitation of sunitinib in plasma samples from a patient receiving daily oral therapy with sunitinib.     

Determination of sulphasalazine and its main metabolite sulphapyridine and 5-aminosalicylic acid in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC-ESI-MS/MS) method has been developed for the simultaneous determination of sulphasalazine (SASP) and its main metabolite sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) with 100 μL of human plasma using dimenhydrinate as the internal standard (I.S.). The API-3000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Protein precipitation process was used to extract SASP, SP, 5-ASA and I.S. from human plasma. The total run time was 9.0 min and the elution of SASP, SP and 5-ASA was at 4.8 min, 2.5 min and 2.0 min, respectively. The separation was achieved with a mobile phase consisting of 0.2% formic acid, 2 mM ammonium acetate in water (mobile phase A) and 0.2% formic acid, 2 mM ammonium acetate in methanol (mobile phase B) by using gradient elution on a XBP Phenyl column (100 mm × 2.1 mm, 5 μm). The developed method was validated in human plasma with a lower limit of quantitation of 10 ng/mL for SASP, SP and 5-ASA, respectively. A linear response function was established for the range of concentrations 10-10,000 ng/mL (r>0.99) for SASP and 10-1000 ng/mL (r>0.99) for SP and 5-ASA. The intra and inter-day precision values for SASP, SP and 5-ASA met the acceptance as per FDA guidelines. SASP, SP and 5-ASA were stable during stability studies, i.e., long term, auto-sampler and freeze/thaw cycles. The method was successfully applied for the evaluation of pharmacokinetics of SASP, SP and 5-ASA after single oral doses of 250 mg SASP to 10 healthy volunteers.     

Analysis of methanol or formic acid in body fluids by headspace solid-phase microextraction and capillary gas chromatography

Methanol and its metabolite formic acid have been found extractable from human whole blood and urine by headspace solid-phase microextraction (SPME) with a Carboxen/polydimethylsiloxane fiber. The headspace SPME for formic acid was carried out after derivatization to methyl formate under acidic conditions. The determinations of both compounds were made by using acetonitrile as internal standard (IS) and capillary gas chromatography (GC) with flame ionization detection. The headspace SPME–GC gave sharp peaks for methanol, methyl formate and I.S.; and low background noises for whole blood and urine samples. Extraction efficiencies were 0.25–1.05% of methanol and 0.38–0.84% formic acid for whole blood and urine. The calibration curves for methanol and formic acid showed excellent linearity in the range of 1.56 to 800 and 1.56 to 500 μg/0.5 ml of whole blood or urine, respectively. The detection limits were 0.1–0.5 μg/0.5 ml for methanol and 0.6 μg/0.5 ml for formic acid for both body fluids. The within-day relative standard deviations in terms of extraction efficiency for both compounds in whole blood and urine samples were not greater than 9.8%. By using the established SPME method, methanol and formic acid were successfully separated and determined in rat blood after oral administration of methanol.     

Human plasma quantification of droperidol and ondansetron used in preventing postoperative nausea and vomiting with a LC/ESI/MS/MS method

An analytical method based upon liquid chromatography coupled to ion trap mass spectrometry (MS) detection with electrospray ionization interface has been developed for the simultaneous identification and quantification of droperidol and ondansetron in human plasma. The two drugs were isolated from 0.5 mL of plasma using a basic liquid-liquid extraction with diethyl ether/heptane (90/10, v/v) and tropisetron and haloperidol as internal standards, with satisfactory extraction recoveries. They were separated on a 5-μm C(18) Highpurity column (150 mm×2.1 mm I.D.) maintained at 30°C. The elution was achieved isocratically with a mobile phase of 2 mM HCOONH(4) pH 3.8 buffer/acetonitrile (60/40, v/v) at a flow rate of 200 μL/min. Data were collected either in full-scan MS mode at m/z 100-450 or in full-scan MS-MS mode, selecting the [M+H] (+) ion at m/z=294.0 for ondansetron, m/z=285.2 for tropisetron, m/z=380.0 for droperidol and m/z=376.0 for haloperidol. The most intense daughter ion of ondansetron (m/z=212.0) and droperidol (m/z=194.0) were used for quantification. Retention times for tropisetron, ondansetron, droperidol and haloperidol were 2.50, 2.61, 3.10 and 4.68 min, respectively. Calibration curves were linear for both compounds in the 0.50-500 ng/mL range. The limits of detection and quantification were 0.10 ng/mL and 0.50 ng/mL, respectively. The intra- and inter-assay precisions were lower than 6.4% and intra- and inter-assay recoveries were in the 97.6-101.9% range for the three 3, 30 and 300 ng/mL concentrations. This method allows simultaneous and rapid measurement of droperidol and ondansetron, which are frequently co-administrated for the prevention of postoperative nausea and vomiting.     

Simultaneous determination of tectorigenin, irigenin and irisflorentin in rat plasma and urine by UHPLC-MS/MS: application to pharmacokinetics

A sensitive and reliable ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) has been developed and validated for the simultaneous determination of three active components, i.e., tectorigenin, irigenin and irisflorentin, in rat plasma and urine after oral administration of Rhizoma Belamcandae extract. Chromatographic separation was achieved on a Zorbax SB-C(18) column (50 mm × 2.1 mm, 1.8 μm; Agilent, USA) with gradient elution using a mobile phase that consisted of acetonitrile - 0.1% formic acid in water at a flow rate of 0.4 mL/min. Detection was performed by a triple-quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via polarity switching between the negative (for tectorigenin and irigenin) and positive (for irisflorentin) ionization modes. The calibration curve was linear over a range of 50-50,000 ng/mL for tectorigenin, 10-5000 ng/mL for irigenin and 0.1-200 ng/mL for irisflorentin, respectively. The intra- and inter-day precisions (RSD %) were within 11.3% for all analytes, whereas the deviation of assay accuracies ranged from -8.7 to +11.1%. All analytes were proven to be stable during all sample storage and analysis procedures. This method was successfully applied to a pharmacokinetic study of the three isoflavones after oral administration of Rhizoma Belamcandae extract to rats.     

HPLC-MS/MS法测定人血浆中草乌甲素的浓度及生物等效性研究(英文)

本文建立了一种快速、高灵敏的HPLC-MS/MS法用于检测人血浆中的草乌甲素浓度。血浆样品采用沃特斯HLB小柱进行固相萃取,汉邦C18色谱柱(150 mm×4.6 mm,5μm)进行分离,流动相为甲醇∶水(85∶15,v/v),水相含10 mmol/L的醋酸铵和0.1%的甲酸。采用ESI源和多反应监测(MRM)的方式进行检测,草乌甲素及内标的反应离子对分别为644.4/584.4和237.2/194.2,草乌甲素血药浓度在0.010～1.0 ng/mL范围内线性关系良好,最低定量限为0.010 ng/mL可以满足口服0.4 mg草乌甲素后血药浓度的检测,日内日间及质控样品精密度及准确度均在允许范围内。本检测方法被成功的应用在中国健康志愿者生物等效性研究中,20名志愿者口服0.4 mg草乌甲素试验制剂和参比制剂后主要药代动力学参数分别如下:Cmax(0.325±0.110),(0.323±0.115)ng/mL;AUC0-16(1.627±0.489),(1.732±0.556)ng.h/mL;AUC0-∞(1.730±0.498),(1.831±0.562)ng.h/mL;t1/2(4.26±0.95),(3.80±0.90)h;Tmax(1.34±0.54),(1.83±0.99)h。     

LC/MS/MS measurement of gentamicin in bovine plasma, urine, milk, and biopsy samples taken from kidneys of standing animals

Methods for the measurement of gentamicin concentration in several bovine tissues were developed and validated. A novel liquid chromatographic (LC) technique employed trifluoroacetic acid in the mobile phase so that all gentamicin components co-eluted. Analytes were ionized by positive-ion pneumatically assisted electrospray and detected by selected reaction monitoring (SRM) with an LC-tandem mass spectrometer (LC/MS/MS). Calibration of plasma and urine samples was based on tobramycin internal standard. Calibration of milk and kidney samples was based on external standard, due to variability of tobramycin response in these matrices. The extraction technique employed treatment with aqueous trichloroacetic acid to both precipitate protein and liberate gentamicin from the matrix. Milk samples had to be defatted by centrifugation prior to extraction. Urine samples were further cleaned up with C-18 solid phase extraction (SPE). These methods were validated for use in several residue depletion studies (reported elsewhere) to monitor the depletion of gentamicin in tissues under various dosing conditions. The plasma method was calibrated from 1 to 5000 ng/mL in two ranges, with a limit of quantitation (LOQ) in the low range calculated at 3.3 ng/mL. The milk method was calibrated from 2.5 to 2500 ng/mL with an LOQ calculated at 4.5 ng/mL. The urine method was designed for use at low levels, and was calibrated from 1 to 100 ng/mL with an LOQ of 3.8 ng/mL. The kidney method was primarily designed for analysis of small samples (approximately 100mg). This method was calibrated from 10 to 50,000 ng/g with an LOQ of 26 ng/g.