Gordon J. Nelson

Liquid Chromatography–Tandem Mass Spectrometry Analysis of Urinary Free Cortisol

For the analysis of urinary free cortisol (UFC), liquid chromatography (LC), LC-mass spectrometry (LC-MS), and liquid chromatography-tandem MS (LC-MS/MS) offer better specificity and accuracy of result than immunoassay-based methods (1). The high specificity of LC-MS/MS provides simplification of sample preparation, reduced costs, increased sample throughput, and a low rate of sample interference. Published approaches to LC-MS/MS analysis of UFC use either sample extraction (1) or direct sample injection (2). The direct methods reduce the labor requirements and decrease the potential for human error. We have developed a direct sample injection method similar to one published by Nassar et al. (2). The primary changes in the proposed method are the use of a guard cartridge for trapping cortisol as part of an online purification strategy and the use of step gradient rather than isocratic LC conditions. Both electrospray ionization (ESI) and atmospheric pressure ionization (APCI) are well suited for LC-MS/MS. We used APCI because preliminary experiments demonstrated a two- to threefold increase in sensitivity compared with ESI. Better sensitivity for cortisol with an APCI interface compared with ESI can be explained by the poor ionization of cortisol in solution. Cortisol and ammonium formate were purchased from Sigma, and d4-cortisol was purchased from Cambridge Isotope Laboratories. Methanol (HPLC grade) was purchased from Fisher Scientific. Patient samples were analyzed for cortisol within 1–3 days after collection. All studies with samples from humans were approved by the Institutional Review Board of the University of Utah. Sample preparation for the method was performed as follows. A 500-μL aliquot of centrifuged urine sample and 100 μL of the working internal standard (IS; d4-cortisol, 0.5 mg/L) were added to autosampler vials, and the vials were vortex-mixed. A PE series 200 HPLC system (Perkin-Elmer Analytical Instruments) was equipped with a Luna C18 column …

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.

Evaluation of reference intervals for methylmalonic acid in plasma/serum and urine ☆

Methylmalonic acid (MMA), an intermediate in the metabolic transformation of propionyl coenzyme A to succinyl coenzyme A catalyzed by vitamin B12 (cobalamin), serves as an indicator of B12 deficiency [1,2]. Vitamin B12 is an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl coenzyme A to succinyl coenzyme A; lack of the vitamin leads to increased concentrations of methylmalonic acid. Vitamin B12 deficiency has been implicated in a spectrum of hematologic, metabolic and neuropsychiatric disorders [3]. Older persons, young children, individuals with malabsorptive or autoimmune disorders and those who follow a strict vegetarian diet are particularly at risk. Both cobalamin and MMA can be measured for diagnosis of cobalamin deficiency. Although serum cobalamin measurement is specific, studies suggest that measurement of serum MMA provides better diagnostic sensitivity, in part because MMA concentrations increase when cobalamin is deficient [4,5]. MMA concentrations are 1000-fold greater than cobalamin concentrations in serum, making MMA easier to measure. Serum methylmalonic acid concentrations are better indicators of tissue vitamin B12 status and MMA is more stable than cobalamin, making MMA measurement clinically more significant. MMA reference intervals (RIs) reported in the literature were developed from studies of relatively small sample size using gas chromatography–mass spectrometry (GC–MS) and vary considerably [6–10]. The purpose of our study was to establish plasma/serum and urine MMA RIs measured using liquid chromatography–tandemmass spectrometry (LC–MS/MS); to determine MMA representative ranges (RRs) [11] by evaluating results for a large, but uncharacterized patient population using a nonparametric statistical approach; and to compare the values to RIs reported in the literature. Non-fasting serum samples were collected from apparently healthy adult subjects (24 females and 16 males) and 128 random urine samples were collected from apparently healthy adults (52 females and 76 males). Samples were tested to measure the MMA concentrations. Analysis was performed using isotope-dilution mass spectrometry following extraction with methyl-tert-butyl ether and derivatization with acidic butanol [12]. The dibutylester of MMA was identified and quantified by LC–MS/MS using a PE series 200 HPLC interfaced to a PE Sciex API 2000 tandem mass spectrometer. The method is based on characteristic fragmentation of the derivatized compound. The protonated molecular (parent) ion, m/z 231, produced by electrospray ionization, is fragmented, and daughter ions at m/z 175 and 119 are monitored. Potential interference due to succinic acid, which has the same molecular weight as MMA, forms a di-ester analogous to MMA, and a parent ion of the same mass-to-charge ratio as MMA, is removed using a mathematical algorithm [13]. The algorithm allows the use of a rapid, high throughput assay in which chromatographic separation

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.