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.

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.

Comparing directly measured versus mathematically calculated free serum 25-hydroxy vitamin D level in children.

INTRODUCTION: 25-Hydroxy vitamin D (25(OH)D) is essential for calcium homeostasis and bone metabolism. The majority of serum 25(OH)D is bound to vitamin D-binding protein (VDBP) (~ 85%) and to albumin (~ 15%), with only a miniscule amount circulating as free 25(OH)D. Free 25(OH)D can be calculated mathematically by Bikle method from the concentrations of total 25(OH)D, VDBP, and albumin or measured directly by ELISA. A direct head-to-head comparison between the two methods has not been done in children. MATERIALS AND METHODS: The objective of the study was to compare the mathematically calculated versus directly measured free 25(OH) vitamin D in children. Serum samples from 74 children (ages 1-19 years) were simultaneously analyzed for total 25(OH)D, serum albumin, VDBP, and free 25(OH)D. Pearson correlation analysis and Bland-Altman plot were used to evaluate agreement between the two methods. RESULTS: The mean age was 9.1 ± 5.1 years, with 61% boys, 76% Caucasians, and 24% African-Americans. The mean ± SD for total 25(OH)D was 38.7 ± 12.8 ng/mL, bioavailable 25(OH)D 3.1 ± 1.1 ng/mL, mathematically calculated free 25(OH)D 8.4 ± 3.2 pg/mL, and directly measured free 25(OH)D 8.9 ± 3.6 pg/mL. Pearson correlation reflected a significant correlation between mathematically calculated and directly measured free 25(OH)D (r = 0.66, p < 0.0005). Bland-Altman plot reflected a tight agreement within a 95% limit of agreement (mean = - 0.026 ± 2SD). CONCLUSIONS: The directly measured and mathematically calculated free 25(OH)D are in close agreement and are interchangeable. Depending on the local availability of instruments and methods, free 25(OH)D can be either directly measured or mathematically calculated.

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).

The Fentanyl Epidemic and Evolution of Fentanyl Analogs in the United States and the European Union.

BACKGROUND: Since 2013, an unprecedented surge in fentanyl overdose deaths has been caused by heroin laced with illicitly produced fentanyl and/or fentanyl analogs (FAs) sold as heroin. The US Drug Enforcement Agency's National Forensic Laboratory Information System reported a >300% increase in fentanyl encounters from 4697 in 2014 to 14440 in 2015. In 2015, the CDC reported 9580 deaths caused by synthetic opioids, primarily fentanyl, a 72% increase from 2014. The European Monitoring Centre for Drugs and Drug Addiction has also encountered several new FAs in the heroin supply. Counterfeit pharmaceuticals containing mixtures of fentanyl and FAs continue to be a poorly recognized worldwide problem despite the WHO classifying several FAs as a serious threat to public health. CONTENT: This review covers the epidemiology of fentanyl abuse and discusses the clinical practice implications of widespread fentanyl abuse. It includes a historical perspective on the illicit FAs that have appeared in the US and European Union and reviews the methods available to identify FAs and emerging technologies useful for identifying previously undescribed analogs. A compilation of structural and mass spectral data on FAs reported thus far is provided. SUMMARY: Fentanyl and FAs have evolved into a global public health threat. It is important to understand the analytical, clinical, and regulatory efforts underway to assist communities affected by the current fentanyl epidemic.

Hydroxycarbamide in children with sickle cell anaemia after first-dose vs. chronic therapy: pharmacokinetics and predictive models for drug exposure.

AIMS: The purposes of this work were to: (1) compare pharmacokinetic (PK) parameters for hydroxycarbamide in children receiving their first dose (HCnew ) vs. those receiving chronic therapy (HCchronic ), (2) assess the external validity of a published PK dosing strategy, and (3) explore the accuracy of dosing strategies based on a limited number of HC measurements. METHODS: Utilizing data from two prospective, multicenter trials of hydroxycarbamide (Pharmacokinetics of Liquid Hydroxyurea in Pediatric Patients with Sickle Cell Anemia; NCT01506544 and Single-Dose (SD) and Steady-State (SS) Pharmacokinetics of Hydroxyurea in Children and Adolescents with Sickle Cell Disease), plasma drug concentration vs. time profiles were evaluated with a model independent approach in the HCnew and HCchronic groups. Various predictive scenarios were analysed to evaluate whether systemic exposure with hydroxycarbamide could be accurately predicted. RESULTS: Absorption of hydroxycarbamide was rapid, variable and dose independent. Dose-normalized peak plasma concentrations and drug exposure (AUC) were higher, and weight-normalized apparent oral clearance was lower in the HCnew group. We assessed a PK-guided dosing strategy along with other predictive scenarios and found that inclusion of plasma samples only slightly improved the accuracy of AUC predictions when compared to a population-based method. CONCLUSIONS: Children naïve to hydroxycarbamide exhibit a different PK profile compared to children receiving chronic therapy. Accuracy of population-based dosing is sufficient to target AUCs in individual patients. Further clearance/bioavailability studies are needed to address the factors responsible for variability in the disposition of hydroxycarbamide.

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.

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.

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.

Technical Note: Significant Positive Bias in Lower Concentrations but Negative Bias in Higher Concentrations in Testosterone Measurement Using Beckman Access Immunoassay Compared to a Liquid Chromatography-Tandem Mass Spectrometry Reference Method.

OBJECTIVE: Testosterone is the principal male sex hormone and is secreted primarily by the testes. In most clinical laboratories testosterone is routinely measured for diagnosis of male hypogonadism or androgen excess in females using FDA approved immunoassays. We compared testosterone values measured by Beckman access immunoassay with those measured by a reference LC-MS/MS method. METHODS: Testosterone was measured in 36 patients using left over serum or plasma specimens by both Beckman immunoassay on the DXI 800 analyzer and a reference LC-MS/MS method. RESULTS: We observed overall significant negative bias of approximately 31.9 % when testosterone values obtained by the reference LC-MS/MS method were plotted in the x-axis and the corresponding testosterone values using the immunoassay in the y-axis, as regression equation was y=0.6887x+38.81 (n=36). The corresponding Deming regression was y=0.6639x+34.7163. However, in eight specimens with low testosterone concentrations, immunoassays significantly overestimated testosterone concentrations. CONCLUSIONS: Immunoassays may underestimate the true testosterone concentration in males but overestimate in females with low testosterone concentration. Therefore, for diagnosis of hypogonadism in males and androgen excess in females, testosterone values obtained by Beckman Access immunoassay on the DXI 800 analyzer should be interpreted with caution.

A simple isotope dilution electrospray ionization tandem mass spectrometry method for the determination of free phenytoin.

BACKGROUND: Phenytoin (diphenylhydantoin) is an anticonvulsant drug frequently prescribed for the treatment of many types of seizures. Because the drug is highly protein bound (90%-95%) and many conditions can displace the drug from proteins, the measurement of free phenytoin is warranted. Due to the unavailability of free phenytoin assays in many chemistry analyzers or limitations of immunoassays, chromatographic methods such as liquid chromatography-tandem mass spectrometry (LC-MS-MS) are preferred for the assay of free phenytoin. METHODS: The sample preparation involved ultrafiltration of serum or plasma to separate free phenytoin. Acetonitrile containing internal standard, phenytoin-d10, was added to the ultrafiltrate. The samples were centrifuged, and supernatants were injected into an LC-MS-MS involving reverse phase Ultra BiPh 5-µm × 50 × 2.1-mm analytical column, and mobile phases, water and methanol containing 0.1% formic acid. The mass/charge (m/z) transitions were as follows: phenytoin -253.0 > 182.2 and 253.0 > 104.00; phenytoin-d10 -263.2 > 192.12. RESULTS: Linearity of the method ranged from 0.1 to 4.0 µg/mL. Within-run and between-run imprecision values were <5% and <10%, respectively. The samples were stable for 2 weeks at 4°C and 4 weeks at -20°C. The method compared well with the laborious liquid-liquid extraction method and did not show any significant ion suppression or enhancement. CONCLUSIONS: A simple LC-MS-MS method was developed for the assay of free phenytoin. The method does not require laborious liquid-liquid or solid-phase extraction. The method has high analytical sensitivity, low imprecision, and a wide analytical measurement range.

Quantitation of Mycophenolic Acid and Mycophenolic Acid Glucuronide in Serum or Plasma by LC-MS/MS.

Mycophenolic acid (MPA) is an immunosuppressant that is used as an adjunct therapy in renal, liver, and heart transplantation. Due to its narrow therapeutic range, monitoring MPA levels is essential to avoid toxicity and organ rejection. Although immunoassays are available for the determination of MPA, mass spectrometry methods are preferred due to their higher specificity. Herein, we describe a liquid chromatography tandem mass spectrometry (LC-MS/MS) method utilizing positive ionization electrospray and multiple reaction monitoring (MRM) for the quantification of MPA levels and its conjugate, MPA glucuronide (MPAG). Blood collected in a plain, EDTA, or heparin-containing tube is centrifuged to separate the serum or plasma. Proteins are precipitated using a zinc sulfate solution and acetonitrile containing deuterated internal standards (MPA-d3 and MPAG-d3). The resulting protein-free supernatant is injected into the LC-MS/MS system for analysis. The chromatography involves the use of a C18 column and ammonium acetate/water/formic acid and ammonium acetate/methanol/formic acid mobile phases. Quantification of MPA and MPAG levels is achieved by comparing the MRM peak area ratios of analytes and internal standards, consisting of specific precursor/product pairs, with those of calibrators at various concentrations. Calibration curves are constructed from the MRM peak area ratios of calibrators and internal standards versus concentration. © 2023 Wiley Periodicals LLC. Basic Protocol: Quantitation of mycophenolic acid and mycophenolic acid glucuronide in serum or plasma by LC-MS/MS.

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.

3-Hydroxyisobutyrate aciduria and mutations in the ALDH6A1 gene coding for methylmalonate semialdehyde dehydrogenase.

3-hydroxyisobutyric aciduria is an organic aciduria with a poorly understood biochemical basis. It has previously been assumed that deficiency of 3-hydroxyisobutyrate dehydrogenase (HIBADH) in the valine catabolic pathway is the underlying enzyme defect, but more recent evidence makes it likely that individuals with 3-hydroxyisobutyryic aciduria represent a heterogeneous group with different underlying mechanisms, including respiratory chain defects or deficiency of methylmalonate semialdehyde dehydrogenase. However, to date methylmalonate semialdehyde dehydrogenase deficiency has only been demonstrated at the gene level for a single individual. We present two unrelated patients who presented with developmental delay and increased urinary concentrations of 3-hydroxyisobutyric acid. Both children were products of consanguineous unions and were of European or Pakistani descent. One patient developed a febrile illness and subsequently died from a hepatoencephalopathy at 2 years of age. Further studies were initiated and included tests of the HIBADH enzyme in fibroblast homogenates, which yielded normal activities. Sequencing of the ALDH6A1 gene (encoding methylmalonate semialdehyde dehydrogenase) suggested homozygosity for the missense mutation c.785 C > A (S262Y) in exon 7 which was not found in 210 control alleles. Mutation analysis of the ALDH6A1 gene of the second patient confirmed the presence of a different missense mutation, c.184 C > T (P62S), which was also identified in 1/530 control chromosomes. Both mutations affect highly evolutionarily conserved amino acids of the methylmalonate semialdehyde dehydrogenase protein. Mutation analysis in the ALDH6A1 gene can reveal a cause of 3-hydroxyisobutyric aciduria, which may present with only slightly increased urinary levels of 3-hydroxyisobutyric acid, if a patient is metabolically stable.

A simple, rapid atmospheric pressure chemical ionization liquid chromatography tandem mass spectrometry method for the determination of 25-hydroxyvitamin D2 and D3.

Vitamin D plays a vital role not only in bone health but also in pathophysiology of many other body functions. In recent years, there has been significant increase in testing of 25-hydroxyvitamin D (25-OH vitamin D), a marker of vitamin D deficiency. The most commonly used methods for the measurement of 25-OH vitamin D are immunoassays and liquid chromatography tandem mass spectrometry (LC-MS-MS). Since immunoassays suffer from inaccuracies and interferences, LC-MS-MS is a preferred method. In LC-MS-MS methods, 25-OH vitamin D is extracted from serum or plasma by solid-phase or liquid-phase extraction. Because these extraction methods are time consuming, we developed an easy method that uses simple protein precipitation followed by injection of the supernatant to LC-MS-MS. Several mass-to-charge (m/z) ratio transitions, including commonly used transitions based on water loss, were evaluated and several tube types were tested. The optimal transitions for 25-OH vitamin D2 and D3 were 395.5 > 269.5 and 383.4 > 257.3, respectively. The reportable range of the method was 1-100 ng/mL, and repeatability (within-run) and within-laboratory imprecision were <4% and <6%, respectively. The method agreed well with the solid-phase extraction methods.

Introduction to Mass Spectrometry for Bimolecular Analysis in a Clinical Laboratory.

Mass spectrometry is a technique that identifies analytes based on mass-to-charge (m/z) ratio and structural fragments. Although this technique has been used in research and specialized clinical laboratories for decades, only in recent years has mass spectrometry become popular in routine clinical laboratories. Mass spectrometry, especially when coupled with gas chromatography or liquid chromatography, provides very specific and often sensitive analysis of many analytes. Other advantages of mass spectrometry include simultaneous analysis of multiple analytes (>100) and generally limited requirement for specialized reagents. Commonly measured analytes by mass spectrometry include metabolites, drugs, hormones, and proteins.

Tandem Mass Spectrometry for the Analysis of Plasma/Serum Acylcarnitines for the Diagnosis of Certain Organic Acidurias and Fatty Acid Oxidation Disorders.

Acylcarnitines are formed in the mitochondria by esterification between carnitine and acyl-CoAs. This occurs enzymatically via carnitine acyltransferases. Specific acylcarnitines accumulate as a result of various organic acidurias and fatty acid oxidation disorders, and, thus, acylcarnitines profiles are used for the diagnosis of these disorders. Acylcarnitines monitoring can also be used for the follow-up of patients with these disorders. Tandem mass spectrometry (MS/MS) is the most commonly used method for the analysis of acylcarnitines. An MS/MS method for the quantification of a number of acylcarnitines is described. The method involves butylation of acylcarnitines using acidified butanol. Butylated acylcarnitines are analyzed using flow injection and precursor ion scan. Multiple-reaction monitoring (MRM) is used for the analysis of low-molecular-weight acylcarnitines.