LC-MS/MS Method for High-Throughput Analysis of Methylmalonic Acid in Serum, Plasma, and Urine: Method for Analyzing Isomers Without Chromatographic Separation.

Measurement of methylmalonic acid (MMA) plays an important role in the diagnosis of vitamin B12 deficiency. Vitamin B12 is an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA (SA-CoA), and the lack of vitamin B12 leads to elevated concentrations of MMA. Measurement of MMA in biological samples is complicated because of the presence of succinic acid (SA), isomer of MMA. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for MMA. The method utilizes derivatization and positive ion mode ionization, which is specific to polycarboxylic acids (MMA and SA are dicarboxylic acids), while derivatives of monocarboxylic acids at these conditions are not ionizable and not detectable. The only organic acid, other than MMA, that is detected in this method is SA. The described method does not require chromatographic resolution of the peaks of MMA and SA; quantitative measurement of MMA is performed using a deconvolution algorithm, which mathematically resolves signal corresponding to MMA, from the combined signal of MMA/SA. Because of the high selectivity of detection, this method utilizes isocratic chromatographic separation; reconditioning and re-equilibration of the chromatographic column between injections is unnecessary. The above features allow high-throughput analysis of MMA with injection-to-injection cycle time of approximately 1 minute.

Simultaneous determination of codeine, morphine, hydrocodone, hydromorphone, oxycodone, and 6-acetylmorphine in urine, serum, plasma, whole blood, and meconium by LC-MS-MS.

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for simultaneous analysis of six major opiates in urine, serum, plasma, whole blood, and meconium is described. The six opiates included are codeine, morphine, hydrocodone, hydromorphone, oxycodone, and 6-acetylmorphine (6-AM). The method was compared to an in-house gas chromatography (GC)-MS method and an LC-MS-MS method performed by another laboratory. The sample preparation time was decreased by eliminating the glucuronide hydrolysis and derivatization required for GC-MS analysis, as well as by adapting the solid-phase extraction to elute directly into autosampler vials. These improvements illustrate the advantages of an LC-MS-MS method over a GC-MS method for opiates. The structural similarity of these six opiates and others in the opiate class causes a high potential for interference and false-positive results. Twelve opiate analogues and metabolites were evaluated for interference. The potential for interference was reduced by altering the MRM transitions chosen for the six opiates. The increased specificity of LC-MS-MS decreased the interference rate in urine to 3.9% compared to 13.6% on the in-house GC-MS method. The rate of positivity for 6-AM in meconium is described for the first time. In urine, 11.0% of morphine positive specimens were also positive for 6-AM compared to 8.3% in serum/plasma and 0.9% in meconium. Although 6-AM is infrequent in meconium, it provides a definitive proof of illegal heroin abuse by the pregnant mother. This method has been routinely used in our laboratory over the last 6 months on more than 1500 patient specimens.

High-Throughput Analysis of Methylmalonic Acid in Serum, Plasma, and Urine by LC-MS/MS. Method for Analyzing Isomers Without Chromatographic Separation.

Measurement of methylmalonic acid (MMA) plays an important role in the diagnosis of vitamin B12 deficiency. Vitamin B12 is an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA (SA-CoA), and the lack of vitamin B12 leads to elevated concentrations of MMA. Presence of succinic acid (SA) complicates the analysis because mass spectra of MMA and SA are indistinguishable, when analyzed in negative ion mode and the peaks are difficult to resolve chromatographically. We developed a method for the selective analysis of MMA that exploits the significant difference in fragmentation patterns of di-butyl derivatives of the isomers MMA and SA in a tandem mass spectrometer when analyzed in positive ion mode. Tandem mass spectra of di-butyl derivatives of MMA and SA are very distinct; this allows selective analysis of MMA in the presence of SA. The instrumental analysis is performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive ion mode, which is, in combination with selective extraction of acidic compounds, is highly selective for organic acids with multiple carboxyl groups (dicarboxylic, tricarboxylic, etc.). In this method organic acids with a single carboxyl group are virtually undetectable in the mass spectrometer; the only organic acid, other than MMA, that is detected by this method is its isomer, SA. Quantitative measurement of MMA in this method is performed using a deconvolution algorithm, which mathematically resolves the signal corresponding to MMA and does not require chromatographic resolution of the MMA and SA peaks. Because of its high selectivity, the method utilizes isocratic chromatographic separation; reconditioning and re-equilibration of the chromatographic column between injections is unnecessary. The above features of the method allow high-throughput analysis of MMA with analysis cycle time of 1 min.

Assessing analytical specificity in quantitative analysis using tandem mass spectrometry.

OBJECTIVES: The necessity of confirmation of compound identity in quantitative analysis is well recognized for methods utilizing single mass spectrometry detection but is not commonly addressed for applications utilizing multiple-stage mass spectrometry (MSn). For MSn detection, no commonly accepted rules for assessment of analytical specificity in quantitative analyses have been established to date. METHODS: To assure compound identity, we evaluated approaches based on monitoring multiple mass transitions of a target compound followed by comparison of the branching ratios of the mass transitions. RESULTS: Monitoring multiple mass transitions along with evaluation of the ratio of their relative intensities allows the analyst to distinguish the target analyte from interferences in quantitative analysis. The strategy and the acceptance criteria are compound and method specific and should be established during the method development and validation. CONCLUSIONS: The certainty of analyte identity is very important in quantitative analysis using MSn detection; methods to verify analyte identity should be used in all critical applications.

Simultaneous analysis of the Delta9-THC metabolites 11-nor-9-carboxy-Delta9-THC and 11-hydroxy-Delta9-THC in meconium by GC-MS.

Neonates that are exposed to cannabinoids in utero may have characteristic physical and mental developmental problems throughout their lives. The early identification of exposed neonates allows early intervention and anticipation of potential problems. Testing meconium detects maternal marijuana use over the last four months of gestation, providing a better drug exposure marker than urine. However, the distribution of metabolites in meconium is not identical to urine and analytical methods must be adapted. Both the major urine metabolite, 11-nor-9-carboxy-Delta9-tetrahydrocannabinol (9-carboxy-THC), and a minor urine metabolite, 11-hydroxy-Delta9-tetrahydrocannabinol (11-hydroxy-THC), are common in meconium. Currently published methods to extract these two metabolites for instrumental analysis are time-consuming and laborious, often involving the preparation of two fractions. This study describes a simple solid-phase extraction method and an optimized hydrolysis method that allow the preparation and analysis of both metabolites in a single extract. The limit of detection by this extraction method was 5 ng/g for both metabolites with an analytical measurement range from 10 to 500 ng/g. The recovery at 100 ng/g was greater than 62% for both analytes. The analysis of 246 cannabinoid screen positive specimens illustrated the importance of including the 11-hydroxy-THC in a meconium marijuana confirmation: 16 specimens confirmed positive for 11-hydroxy-THC only, resulting in a 6.5% increase in the positivity rate compared to 9-carboxy-THC alone.

Pseudo-Cushing syndrome caused by fenofibrate interference with urinary cortisol assayed by high-performance liquid chromatography.

Urinary free cortisol (UFC) excretion over 24 h reflects the production rate of cortisol and is used commonly in the diagnosis of Cushing syndrome. We report on two patients evaluated for Cushing syndrome who had elevated UFC when analyzed by HPLC but normal values for the analysis performed by RIA and HPLC-mass spectrometry/mass spectrometry (HPLC-MS/MS). Other laboratory testing was inconsistent with the diagnosis of Cushing syndrome and raised doubts about the diagnosis. We identified a probable cause of analytical interference as coming from fenofibrate (Tricor), medication taken by the patients. Fenofibrate peak overlapped with the HPLC peak of cortisol and produced an MS/MS transition overlapping the major transition of cortisol. A second MS/MS transition was free from interference. In summary, fenofibrate administration may cause false elevation of UFC values determined by HPLC or HPLC-MS/MS in patients evaluated for Cushing syndrome. An HPLC-MS/MS method using multiple mass transitions, rather than a single transition, allows accurate quantitation of urinary cortisol in patients taking fenofibrate.

Dissociation of individual isotopic peaks: predicting isotopic distributions of product ions in MSn.

Traditional practice in tandem mass spectrometry is to select the mono-isotopic ion for dissociation. However, high molecular weight compounds often have weak mono-isotopic peaks, which limit that approach. Furthermore, the traditional approach does not take advantage of the very rich store of information available in the isotopic patterns from the dissociation of individual non-mono-isotopic peaks. Interpretation of these isotopic patterns requires a theory capable of predicting the patterns. However, a general theory for the prediction of these patterns has been lacking. This paper shows that the patterns can be obtained from a certain vector product, the outer product, of the full isotopic distribution of the product ion with the full isotopic distribution of the complementary product. Unlike previous approaches, the method is applicable to systems of arbitrary isotopic complexity. The patterns are potentially useful for elucidation of dissociation pathways, elemental composition, and chemical structure. The paper presents several applications of the theory.

Simultaneous quantitative analysis of isobars by tandem mass spectrometry from unresolved chromatographic peaks.

A method was developed for the simultaneous quantitation of isobars from unresolved chromatographic peaks. The method is based on differences in branching ratios of ion abundances in their tandem mass spectra and an assumption that the product ion mass spectra of a mixture can be considered as a linear combination of the spectra of individual constituents. We present analytical equations and a matrix-based approach for deconvoluting the concentration of individual components from the total peak intensity for two and three isobars and also a matrix-based generalization to any number of compounds. The feasibility of the simultaneous analysis of mixtures containing two compounds was assessed. The approach was evaluated for the analysis of structural isomers of methylmalonic and succinic acids in human plasma and urine samples for a group of 270 samples. The linear regression equation, standard error and correlation coefficient for the agreement with a traditional method utilizing chromatographic separation of the isomers were y = 0.999x - 0.005, 0.024 micro mol l(-1), and 0.985, respectively. The utility of a spectral contrast angle as a predictor of analysis feasibility was evaluated.

Evaluation of the NexScreen and DrugCheck Waive RT urine drug detection cups.

Urine drug testing is an important tool that is commonly used to assess patient compliance with prescription regimens. Point-of-collection immunoassay devices allow for timely availability of laboratory test results to guide therapy during the same office visit. Two waived immunoassay-based urine drug screen cups were evaluated in this study. The NexScreen cup and the DrugCheck Waive RT cup claim to detect 10-12 drug classes of commonly used and/or abused drugs. This study included a sensitivity and precision challenge with 4-6 replicates at concentrations 0-150% of the manufacture's claimed cutoff, using drug-free urine spiked with purified reference standards. The stability of test results was evaluated by reading the results at intervals between five and 1,440 min. Specificity was evaluated by parallel comparison of pooled patients' specimens, representing 56 patients and 41 known drug compounds. When comparing results to validated liquid chromatography-mass spectrometry results, false positives were observed in the NexScreen cups for benzodiazepine, methamphetamine, methadone, opiates and tricyclic antidepressant tests, but there were no false negatives. The DrugCheck Waive RT cups showed false negative results for barbiturates and opiates, but no false positives. Overall, the NexScreen cup demonstrated better sensitivity than claimed, whereas the sensitivity of the DrugCheck Waive RT cup did not meet claims.