Evaluation of a Delta-9-Tetrahydrocannabinol Carboxylic Acid (Δ9-THC-COOH) Immunoassay and a Gas Chromatography-Mass Spectrometry (GC-MS) Method for the Detection of Delta-8-Tetrahydrocannabinol Carboxylic Acid (Δ8-THC-COOH).

BACKGROUND: Delta-8 tetrahydrocannabinol (Δ8-THC) is a naturally occurring or synthetically prepared cannabinoid that elicits psychological and physiological experiences commonly reported for its more infamous isomer, delta-9 tetrahydrocannabinol (Δ9-THC). Unlike Δ9-THC, Δ8-THC products are generally legal under federal law and there has been a rise in their usage. One of the main targets for detection and quantitation of Δ9-THC is its inactive metabolite, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THC-COOH). METHODS: This study evaluated the ability of the currently used Δ9-THC-COOH immunoassay and gas chromatography-mass spectrometry (GC-MS) methods to detect 11-nor-9-carboxy-Δ8-tetrahydrocannabinol (Δ8-THC-COOH) and distinguish it from Δ9-THC-COOH. RESULTS: The EMIT II Plus® Cannabinoid immunoassay for Δ9-THC-COOH with a cutoff of 20 ng/mL showed positive results for Δ8-THC-COOH with concentrations of 30 ng/mL or higher. Although many of the ion fragments generated by mass spectrometry were found to overlap between the 2 compounds, the GC-MS method presently used to quantify Δ9-THC-COOH separated the 2 compounds sufficiently to identify them independently by relative retention time. CONCLUSION: Current immunoassays and GC-MS methods should be evaluated for the ability to detect and distinguish the presence of Δ8-THC-COOH.

The effect of burosumab on intact and C-terminal FGF23 measurements.

OBJECTIVE: To investigate the effect of CRYSVITA® (burosumab-twza) on FGF23 measurements in an intact and a C-terminal immunoassay. METHODS: An intact serum FGF23 (MedFrontier) and a C-terminal plasma FGF23 assay (Immutopics) were used. Serum/plasma pools were spiked to span the burosumab therapeutic range (1.4-11.3 µg/ml) and FGF23 recovery was assessed. Patient serum and plasma samples obtained pre and post-burosumab treatment were evaluated on both assays and compared with corresponding phosphorus measurements RESULTS: Spiking burosumab (1.4-11.3 µg/ml) into sample pools resulted in a dose-dependent negative analytical interference on intact FGF23 measurements and no significant interference for C-terminal FGF23 measurements. However, more than a 500-fold median increase (post- vs. pre-burosumab administration) in in vivo FGF23 concentrations were observed by both assays. CONCLUSIONS: Therapeutic concentrations of burosumab result in a negative analytical interference of the intact, but not the C-terminal FGF23 immunoassay. Despite this in vitro analytical interference in the intact assay, relatively large elevations of both intact FGF23 and C-terminal FGF23 measurements were observed in vivo following burosumab administration. Following burosumab administration, FGF23 measurements must be interpreted within the clinical context of the patient and other relevant biomarker results. SUMMARY: This article describes a negative analytical interference by burosumab in an intact FGF23 immunoassay. The recovery of C-terminal FGF23 is not significantly affected by the presence of burosumab. In vivo, both assays demonstrate extreme FGF23 elevations in the presence of the drug. Furthermore, the measurement of FGF23 blocked by burosumab is not clinically useful regarding hypophosphataemia.

Quantitation of Neuroblastoma Markers Homovanillic Acid (HVA) and Vanillylmandelic Acid (VMA) in Urine by Gas Chromatography-Mass Spectrometry (GC/MS).

Neuroblastoma and other neural crest tumors can be characterized by the increased production and excretion of catecholamines and their metabolites. Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are important catecholamine metabolites that can be measured to provide relatively rapid laboratory diagnosis and clinical follow-up of neuroblastoma. We present a procedure to quantify HVA and VMA in urine samples which have been diluted to a creatinine concentration of 2 mg/dL. Diluted samples are spiked with deuterated internal standards, acidified, and extracted with an organic solvent. A bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and pyridine mixture is added to the dried extract to create trimethylsilyl derivatives of HVA and VMA. The derivatized compounds are measured using gas chromatography-mass spectrometry (GC/MS).

Screening of Organic Acidurias by Gas Chromatography-Mass Spectrometry (GC-MS).

Organic acidurias or acidemias are a group of diverse disorders caused by decreased or diminished activity of specific enzyme or transporter involved in the metabolism of amino acids, carbohydrates, fatty acids, and nucleic acids. Organic acidurias are generally inherited but may be acquired due to deficiency of certain cofactors or vitamins. As clinical symptoms are of nonspecific nature, definitive diagnosis of organic aciduria requires measurement of organic acids in urine or blood and sometimes enzyme activity in the cells. Gas chromatography-mass spectrometry (GC-MS) is a commonly used method for screening of organic acidurias.GC-MS procedure described here involves the use of urine volume that contains 1 µmole (113 µg) of creatinine. Internal standards (tropic and 2-ketocaproic acids) are added to the samples, followed by treatment with hydroxylamine to form oxime derivatives of the ketoacids. The mixture is then acidified, and organic acids are extracted in ethyl acetate. The organic extract is concentrated to dryness, and the residue is treated with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)/trimethylchlorosilane (TMCS)/pyridine to form the trimethylsilyl (TMS) derivatives of the organic acids. The derivatized extract is then directly injected onto GC-MS for analysis.

Quantification of Tryptophan, Indole, and Indoxyl Sulfate in Urine Using Liquid Chromatography-Tandem Mass Spectrometry.

Having a diverse gut microbiota has been correlated with the short- and long-term success of allogeneic stem cell transplantation. Intestinal bacteria metabolize the amino acid tryptophan to indole. Indole is further oxidized and sulfonated in the liver to 3-indoxyl sulfate (3-IS), which is then excreted in urine. Urinary 3-IS is a potential biomarker for intestinal health and an early predictor of successful stem cell transplantation. We describe a rapid method for quantifying tryptophan, indole, and 3-indoxyl sulfate in urine specimens, in which urine samples are diluted with a formic acid solution and deuterated internal standards, and then injected on LC-MS/MS for analysis.

Peppermint oil effects on the gut microbiome in children with functional abdominal pain.

Peppermint oil (PMO) is effective in the treatment of functional abdominal pain disorders, but its mechanism of action is unclear. Evidence suggests PMO has microbicidal activity. We investigated the effect of three different doses of PMO on gut microbiome composition. Thirty children (7-12 years of age) with functional abdominal pain provided a baseline stool sample prior to randomization to 180, 360, or 540 mg of enteric coated PMO (10 participants per dose). They took their respective dose of PMO (180 mg once, 180 mg twice, or 180 mg thrice daily) for 1 week, after which the stool collection was repeated. Baseline and post-PMO stools were analyzed for microbiome composition. There was no difference in alpha diversity of the gut microbiome between the baseline and post-PMO treatment. Principal coordinate analysis revealed no significant difference in overall bacterial composition between baseline and post-PMO samples, as well as between the PMO dose groups. However, the very low abundant Collinsella genus and three operational taxonomic units (one belonging to Collinsella) were significantly different in samples before and after PMO treatment. The Firmicutes/Bacteroidetes ratio was lower in children who received 540 mg of PMO compared to the 180 mg and 360 mg dose groups (p = 0.04). Network analysis revealed separation between pre- and post-PMO fecal samples with the genus Collinsella driving the post-PMO clusters. PMO administration appeared to impact only low abundance bacteria. The 540 mg PMO dose differentially impacted the Firmicutes/Bacteroidetes ratio. A higher dose and/or longer duration of treatment might yield different results.

Randomised trial: Peppermint oil (menthol) pharmacokinetics in children and effects on gut motility in children with functional abdominal pain.

AIMS: Little is known regarding the pharmacokinetics and pharmacodynamics of menthol, the active ingredient in peppermint oil (PMO). Our aim was to investigate the pharmacokinetics of menthol at 3 dose levels in children and determine their effects on gut motility and transit. METHODS: Thirty children ages 7-12 years with functional abdominal pain underwent wireless motility capsule (WMC) testing. Approximately 1 week later they were randomized to 180, 360 or 540 mg of enteric coated PMO (10 participants per dose). Menthol pharmacokinetics were determined via blood sampling over 24 hours. They then took their respective dose of PMO (180 mg once, 180 mg twice or 180 mg thrice daily) for 1 week during which time the WMC test was repeated. RESULTS: Evaluable area under the plasma concentration vs. time curve (AUClast ) data were available in 29 of 30 participants. A direct linear relationship (apparent dose-proportionality for systemic menthol exposure) was observed between PMO dose and menthol systemic exposure with mean elimination half-life 2.1, 3.5 and 4.6 hours for the 180, 360 and 540 mg doses, respectively. WMC technical issues precluded complete motility data in all participants. Colonic transit time was inversely related to AUClast (P = .003); transit time in other regions was not affected. In contrast, stomach, small bowel and whole gut (but not colonic) contractility positively correlated with menthol AUClast (P < .05). CONCLUSION: Pharmacokinetics and pharmacodynamics of menthol derived from PMO demonstrated apparent dose-proportionality. A higher dose of PMO may be needed to achieve maximal gut response. www.clinicaltrials.gov NCT03295747.

Measuring hydroxyurea adherence by pharmacy and laboratory data compared with video observation in children with sickle cell disease.

BACKGROUND: Hydroxyurea nonadherence is common among children with sickle cell disease (SCD), but it is unclear if current adherence measures are valid compared with video directly observed therapy (VDOT), a reference method. The objectives were to evaluate if hydroxyurea adherence by pharmacy records, urine assay, mean corpuscular volume (MCV), and/or fetal hemoglobin (HbF) correlated with and was sensitive and specific compared with VDOT. METHODS: This was a cross-sectional analysis of adherence data from 34 children with SCD on a single-arm, six-month hydroxyurea adherence study. Spearman correlation coefficient compared participants' adherence by pharmacy records, MCV, and HbF to adherence by VDOT. The sensitivity and specificity of ≥80% adherence by pharmacy records, two urine samples with hydroxyurea, MCV ≥100 fl/L, and HbF ≥20% compared with ≥80% VDOT adherence were also calculated. RESULTS: Median pharmacy and VDOT adherence rates were similar (87.8% vs 88.1%, P = 0.75) and mildly correlated (rs  = 0.45; P = 0.008) but the sensitivity of ≥80% adherence by pharmacy records was 72.7% and specificity was 45.5%. MCV (rs  = -0.02, P = 0.92) and HbF (rs  = -0.2, P = 0.33) did not significantly correlate with VDOT adherence. Sensitivity and specificity were 83.3% and 33.3% for having two urine samples with hydroxyurea, 35% and 71.4% for MCV ≥100 fl/L, and 75% and 0% for HbF ≥20%, respectively. CONCLUSIONS: Commonly used tools to measure hydroxyurea adherence may not correlate with or be valid compared with video adherence. Future studies to refine these measures are needed to effectively target adherence interventions to children with SCD who have the potential to benefit. (ClinicalTrials.gov NCT02578017).

Association of Biotin Ingestion With Performance of Hormone and Nonhormone Assays in Healthy Adults.

Importance: Biotinylated antibodies and analogues, with their strong binding to streptavidin, are used in many clinical laboratory tests. Excess biotin in blood due to supplemental biotin ingestion may affect biotin-streptavidin binding, leading to potential clinical misinterpretation. However, the degree of interference remains undefined in healthy adults. Objective: To assess performance of specific biotinylated immunoassays after 7 days of ingesting 10 mg/d of biotin, a dose common in over-the-counter supplements for healthy adults. Design, Setting, and Participants: Nonrandomized crossover trial involving 6 healthy adults who were treated at an academic medical center research laboratory. Exposure: Administration of 10 mg/d of biotin supplementation for 7 days. Main Outcomes and Measures: Analyte concentrations were compared with baseline (day 0) measures on the seventh day of biotin treatment and 7 days after treatment had stopped (day 14). The 11 analytes included 9 hormones (ie, thyroid-stimulating hormone, total thyroxine, total triiodothyronine, free thyroxine, free triiodothyronine, parathyroid hormone, prolactin, N-terminal pro-brain natriuretic peptide, 25-hydroxyvitamin D) and 2 nonhormones (prostate-specific antigen and ferritin). A total of 37 immunoassays for the 11 analytes were evaluated on 4 diagnostic systems, including 23 assays that incorporated biotin and streptavidin components and 14 assays that did not include biotin and streptavidin components and served as negative controls. Results: Among the 2 women and 4 men (mean age, 38 years [range, 31-45 years]) who took 10 mg/d of biotin for 7 days, biotin ingestion-associated interference was found in 9 of the 23 (39%) biotinylated assays compared with none of the 14 nonbiotinylated assays (P = .007). Results from 5 of 8 biotinylated (63%) competitive immunoassays tested falsely high and results from 4 out of 15 (27%) biotinylated sandwich immunoassays tested falsely low. Conclusions and Relevance: In this preliminary study of 6 healthy adult participants and 11 hormone and nonhormone analytes measured by 37 immunoassays, ingesting 10 mg/d of biotin for 1 week was associated with potentially clinically important assay interference in some but not all biotinylated assays studied. These findings should be considered for patients taking biotin supplements before ordering blood tests or when interpreting results. Trial Registration: clinicaltrials.gov Identifier: NCT03034707.

Do rapid comprehensive urine drug screens change clinical management in children?

CONTEXT: Multiple studies have concluded that urine drug screens rarely change clinical management. The rapid comprehensive urine drug screen (RCUDS) at our institution detects over 300 substances using a combination of EIA and GC/MS and typically takes 2-5 h for completion. OBJECTIVE: We sought to determine whether this RCUDS altered management in the pediatric population. METHODS: All patients >1 month and <18 years of age in which a RCUDS was completed from 1 January 2012 to 31 December 2012 were eligible for the study. Assuming that clinical management would not be altered in at least 90% of cases with a confidence interval of 95%, an alpha error of 5%, we calculated a sample size of 122 cases to ensure adequate study power. Four board-certified medical toxicologists reviewed 160 cases. Cases were assigned to the toxicologists based on a random-number generator. In addition, each toxicologist reviewed 12 random cases from the other three toxicologist's cases to determine inter-rater reliability. All four toxicologists reviewed any case in which a RCUDS was believed to have changed management. RESULTS: A total of 908 RCUDS were performed during the study period, and 160 were selected for study. Mean age was 10.5 years; male = 83, female = 77. Most were ordered from the ED (101/160 = 63%), followed by the inpatient unit (36/160 = 23%), outpatient (14/160 = 9%), and ICU (9/160 = 6%). 111/160 (69%) had a history of ingestion. Of the 160 randomly chosen cases, only three cases were found in which overall clinical management was altered based on the results of the RCUDS. All three cases were children <3 years old with a RCUDS positive for amfetamines. In all the three cases, police, Division of Family Services (DFS), and social work were involved. In no case did the acute clinical management change occurred due to the results of the RCUDS. CONCLUSIONS: The RCUDS rarely changed management in patients at our institution. Further study is warranted.

Design and Testing of an EHR-Integrated, Busulfan Pharmacokinetic Decision Support Tool for the Point-of-Care Clinician.

BACKGROUND: Busulfan demonstrates a narrow therapeutic index for which clinicians routinely employ therapeutic drug monitoring (TDM). However, operationalizing TDM can be fraught with inefficiency. We developed and tested software encoding a clinical decision support tool (DST) that is embedded into our electronic health record (EHR) and designed to streamline the TDM process for our oncology partners. METHODS: Our development strategy was modeled based on the features associated with successful DSTs. An initial Requirements Analysis was performed to characterize tasks, information flow, user needs, and system requirements to enable push/pull from the EHR. Back-end development was coded based on the algorithm used when manually performing busulfan TDM. The code was independently validated in MATLAB using 10,000 simulated patient profiles. A 296-item heuristic checklist was used to guide design of the front-end user interface. Content experts and end-users (n = 28) were recruited to participate in traditional usability testing under an IRB approved protocol. RESULTS: Decision support software was developed to systematically walk the point-of-care clinician through the TDM process. The system is accessed through the EHR which transparently imports all of the requisite patient data. Data are visually inspected and then curve fit using a model-dependent approach. Quantitative goodness-of-fit are converted to single tachometer where "green" alerts the user that the model is strong, "yellow" signals caution and "red" indicates that there may be a problem with the fitting. Override features are embedded to permit application of a model-independent approach where appropriate. Simulations are performed to target a desired exposure or dose as entered by the clinician and the DST pushes the user approved recommendation back into the EHR. Usability testers were highly satisfied with our DST and quickly became proficient with the software. CONCLUSIONS: With early and broad stake-holder engagement we developed a clinical DST for the non-pharmacologist. This tools affords our clinicians the ability to seamlessly transition from patient assessment, to pharmacokinetic modeling and simulation, and subsequent prescription order entry.

Intravenous busulfan dose individualization - impact of modeling approach on dose recommendation.

TDM is intended to limit unintended consequences of drugs with narrow therapeutic indices. However, the application of different sampling strategies and pharmacokinetic approaches results in different dosing recommendations and ostensibly different outcomes. TDM approaches for intravenous busulfan dose individualization employ compartmental or non-compartmental modeling with anywhere from three to seven drug levels. This investigation was designed to examine the differences in dosing recommendations that arise in children (n = 30) when five different TDM approaches were employed. Significant differences in recommended doses between modeling strategies were observed. More importantly, the recommendations were discordant in 13 cases with at least one model recommending a dose adjustment in the opposite direction relative to the remaining models. The mathematical differences introduced by the application of different TDM approaches are not purely academic. Unification of busulfan TDM approaches should be considered to mitigate inconsistently applied dose adjustment, and facilitate comparisons of outcome, between clinical centers.

Quantification of Five Clinically Important Amino Acids by HPLC-Triple TOF™ 5600 Based on Pre-column Double Derivatization Method.

Phenylalanine, tyrosine, glycine, cystine, and phosphoethanolamine are commonly measured amino acids in various physiological fluids to diagnose or follow-up various inborn errors of metabolism. The gold standard method for the amino acids quantitation has been ion exchange chromatography with ninhydrin post-column derivatization. However, this method is very laborious and time consuming. In recent years, liquid-chromatography mass spectrometry is being increasingly used for the assay of amino acids. Pre-column butyl derivatization with reverse phase chromatography has been widely used for mass spectrometry analysis of amino acids. Phosphoethanolamine is not butylated and cannot be measured by this method. Nevertheless, phosphoethanolamine can be dansyl-derivatized using dansyl chloride. We developed a double derivatization method by using butanol and dansyl chloride to derivatize carboxylic and amino groups separately, and then combining the derivatives to simultaneously measure these five amino acids using TOF-MS scan. Stable isotope-labeled internal standards were used.

A Simple Liquid Chromatography Tandem Mass Spectrometry Method for Quantitation of Plasma Busulfan.

Busulfan is an alkylating agent widely used in the ablation of bone marrow cells before hematopoietic stem cell transplant. Due to large intraindividual and interindividual variations, and narrow therapeutic window, therapeutic drug monitoring of busulfan is warranted. A quick and reliable HPLC-MS/MS method was developed for the assay of plasma busulfan. HPLC involved C18 column, and MS/MS was used in electrospray ionization (ESI) positive mode. Quantitation and identification of busulfan was made using various multiple reactions monitoring (MRMs). Isotopic labeled busulfan-d8 was used as the internal standard. The method is linear from 50 to 2500 ng/mL and has with-in run and between-run imprecision of <10 %.

Quantification of 11-Carboxy-Delta-9-Tetrahydrocannabinol (THC-COOH) in Meconium Using Gas Chromatography/Mass Spectrometry (GC/MS).

Maternal substance abuse is an ongoing concern and detecting drug use during pregnancy is an important component of neonatal care when drug abuse is suspected. Meconium is the preferred specimen for drug testing because it is easier to collect than neonatal urine and it provides a much broader time frame of drug exposure. We describe a method for quantifying 11-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in meconium. After adding a labeled internal standard (THC-COOH D9) and acetonitrile, samples are sonicated to release both free and conjugated THC-COOH. The acetonitrile/aqueous layer is removed and mixed with a strong base to hydrolyze the conjugated THC-COOH. The samples are then extracted with an organic solvent mixture as part of a sample "cleanup." The organic solvent layer is discarded and the remaining aqueous sample is acidified. Following extraction with a second organic mixture, the organic layer is removed and concentrated to dryness. The resulting residue is converted to a trimethylsilyl (TMS) derivative and analyzed using gas chromatography/mass spectrometry (GC/MS) in selective ion monitoring (SIM) mode.

Comprehensive Urine Drug Screen by Gas Chromatography/Mass Spectrometry (GC/MS).

Drug screening is an essential component of clinical toxicology laboratory service. Some laboratories use only automated chemistry analyzers for limited screening of drugs of abuse and few other drugs. Other laboratories use a combination of various techniques such as immunoassays, colorimetric tests, and mass spectrometry to provide more detailed comprehensive drug screening. Mass spectrometry, gas or liquid, can screen for hundreds of drugs and is often considered the gold standard for comprehensive drug screening. We describe an efficient and rapid gas chromatography/mass spectrometry (GC/MS) method for comprehensive drug screening in urine which utilizes a liquid-liquid extraction, sample concentration, and analysis by GC/MS.

Determination of Menthol in Plasma and Urine by Gas Chromatography/Mass Spectrometry (GC/MS).

Menthol, a monoterpene, is a principal component of peppermint oil and is used extensively in consumer products as a flavoring aid. It is also commonly used medicinally as a topical skin coolant; to treat inflammation of the mucous membranes, digestive problems, and irritable bowel syndrome (IBS); and in preventing spasms during endoscopy and for its spasmolytic effect on the smooth muscle of the gastrointestinal tract. Menthol has a half life of 3-6 h and is rapidly metabolized to menthol glucuronide which is detectable in urine and serum following menthol use. We describe a method for the determination of total menthol in human plasma and urine using liquid/liquid extraction, gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring mode and menthol-d4 as the internal standard. Controls are prepared with menthol glucuronide and all samples undergo enzymatic hydrolysis for the quantification of total menthol. The method has a linear range of 5-1000 ng/mL, and coefficient of variation <10%.

Systemic exposure to menthol following administration of peppermint oil to paediatric patients.

OBJECTIVE: Peppermint oil (PMO) has been used to treat abdominal ailments dating to ancient Egypt, Greece and Rome. Despite its increasing paediatric use, as in irritable bowel syndrome (IBS) treatment, the pharmacokinetics (PK) of menthol in children given PMO has not been explored. DESIGN AND SETTING: Single-site, exploratory pilot study of menthol PK following a single 187 mg dose of PMO. Subjects with paediatric Rome II defined (IBS; n=6, male and female, 7-15 years of age) were enrolled. Blood samples were obtained before PMO administration and at 10 discrete time points over a 12 h postdose period. Menthol was quantitated from plasma using a validated gas chromatography mass spectrometry technique. Menthol PK parameters were determined using a standard non-compartmental approach. RESULTS: Following a dose of PMO, a substantial lag time (range 1-4 h) was seen in all subjects for the appearance of menthol which in turn, produced a delayed time of peak (Tmax=5.3 ± 2.4 h) plasma concentration (Cmax=698.2 ± 245.4 ng/mL). Tmax and Tlag were significantly more variable than the two exposure parameters; Cmax, mean residence time and total area under the curve (AUC=4039.7 ± 583.8 ng/mL × h) which had a coefficient of variation of <20%. CONCLUSIONS: Delayed appearance of menthol in plasma after oral PMO administration in children is likely a formulation-specific event which, in IBS, could increase intestinal residence time of the active ingredient. Our data also demonstrate the feasibility of using menthol PK in children with IBS to support definitive studies of PMO dose-effect relationships.

Isotope-dilution gas chromatography-mass spectrometry method for the analysis of hydroxyurea.

BACKGROUND: Hydroxyurea is used in the treatment of various malignancies and sickle cell disease. There are limited studies on the pharmacokinetics of hydroxyurea, particularly in pediatric patients. An accurate, precise, and sensitive method is needed to support such studies and to monitor therapeutic adherence. We describe a novel gas chromatography-mass spectrometry (GC-MS) method for the determination of hydroxyurea concentration in plasma using stable labeled hydroxyurea C N2 as an internal standard. METHODS: The method involved an organic extraction followed by the preparation of trimethylsilyl (TMS) derivatives of hydroxyurea for GC-MS selected ion-monitoring analysis. The following mass-to-charge (m/z) ratio ions for silated hydroxyurea and hydroxyurea C N2 were monitored: hydroxyurea-quantitative ion 277, qualifier ions 292 and 249; hydroxyurea C N2-quantitative ion 280, qualifier ion 295. This method was evaluated for reportable range, accuracy, within-run and between-run imprecisions, and limits of quantification. RESULTS: The reportable range for the method was 0.1-100 mcg/mL. All results were accurate within an allowable error of 15%. Within-run and between-run imprecisions were <15%. Samples were stable for at least 4 hours at room temperature, 2 months at -20°C, and 6 months at -70°C, and after 3 freeze/thaw cycles. Extraction efficiency for 1-, 5-, 10-, and 50-mcg/mL samples averaged 2.2%, 1.8%, 1.6%, and 1.4%, respectively. CONCLUSIONS: The isotope-dilution GC-MS method for analysis of hydroxyurea described here is accurate, sensitive, precise, and robust. Its characteristics make the method suitable for supporting pharmacokinetic studies and/or clinical therapeutic monitoring.