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Evaluation of a Simple Method for Methylmalonic Acid Analysis in Human Plasma by LC-MS/MS

Abstract

A new LC-MS/MS method for methylmalonic acid (MMA) analysis was developed that provides complete chromatographic resolution from isobaric succinic acid in plasma samples. Excellent chromatographic results were obtained from the direct injection of a protein crash sample supernatant onto a Force C18 column, providing a much simpler sample preparation compared to typical methods.

Introduction

Vitamin B12 deficiency can manifest clinically in a wide variety of physical and behavioral signs and symptoms, but methylmalonic acid (MMA) can be used as a specific biomarker for diagnosis. In the metabolic cycle for energy production, vitamin B12 promotes the conversion of methylmalonyl CoA to succinyl CoA. If there is not enough B12 available, blood levels of MMA begin to rise. High levels of MMA can also result from the metabolic disorder methylmalonic acidemia, which causes the inability to properly digest specific fats and proteins. Therefore, elevated levels of MMA can be used to diagnose functional vitamin B12 deficiency as well as methylmalonic acidemia.

The MMA test typically requires extensive sample pre-treatment incorporating liquid-liquid extraction, derivatization, solvent evaporation, and/or SPE. Additionally, chromatographic resolution can be difficult to achieve between MMA and its naturally occurring isomer, succinic acid. Herein, we present a simple sample preparation method—without derivatization—that allows for the direct injection of protein crash supernatant while still maintaining resolution between MMA and succinic acid in a 5-minute cycle time.

Figure 1: Structures of Methylmalonic Acid and Succinic Acid.

Structures of Methylmalonic Acid and Succinic Acid

Experimental

Calibration Standards and Quality Control Samples

Double charcoal stripped human plasma contains endogenous levels of methylmalonic acid; therefore, a surrogate matrix was created by fortifying SeraFlx BIOMATRIX (Cerilliant) at 1000 ng/mL with succinic acid. The surrogate matrix was then spiked with MMA to prepare calibration standards and QC samples. The calibration range was 10–500 ng/mL and surrogate matrix QC samples were prepared at 10, 20, 150, and 400 ng/mL.

The amount of endogenous methylmalonic acid in the double charcoal stripped human plasma was determined to be 13 ng/mL using the calibration standards prepared in the surrogate matrix. Additional QC samples were prepared in double charcoal stripped human plasma by fortifying at the same levels as the surrogate matrix and then quantitatively accounting for the additional endogenous MMA level. The QC samples in human plasma contained combined (endogenous and fortified) MMA levels of 23, 33, 163, and 413 ng/mL.

Internal standard MMA-D3 was prepared at 2,500 ng/mL in water; 5 µL was then added to each sample, and samples were vortexed prior to protein precipitation.

Sample Preparation

Protein precipitation was performed by aliquoting 300 µL of 0.5% formic acid in methanol into 100 µL of sample. The sample was then vortexed for 10 seconds at 3000 rpm followed by centrifugation at 4000 rpm for 10 minutes at 10 °C. The supernatant (250 µL) was then filtered using a Thomson SINGLE StEP standard filter vial (0.2 µm PVDF membrane, Restek cat.# 25895).

Analytical column:Force C18 3 µm, 100 mm x 3.0 mm (cat.# 963431E)
Guard column:Force C18 EXP guard column cartridge (cat.# 963450253)
Mobile phase A:Water, 0.5% formic acid
Mobile phase B:Methanol, 0.5% formic acid
GradientTime (min)%B
0.005
0.505
3.0095
3.015
5.005
Flow rate:0.7 mL/min
Injection volume:3 µL
Column temp.: 35 °C
Sample temp.: 10 °C
Ion mode:Negative ESI

Table I: Analyte Transitions for Methylmalonic Acid Analysis.

Compound

Precursor Ion

Product Ion

MMA

117.3

73.1

MMA-D3

120.2

76.2


Results and Discussion

Sample Preparation Evaluation

To ensure that the supernatant was compatible with direct analysis, an initial solvent evaluation was conducted. Trichloroacetic acid (TCA) was initially investigated for use as the precipitating solvent. While using a small volume of 40% TCA (w/v) (25 µL per 100 µL of sample) effectively precipitated proteins, it resulted in no signal for methylmalonic acid, despite dilution with 300 µL of 0.5% formic acid in water prior to analysis (Figure 2). It is speculated that the TCA present in the final extract is not only responsible for the improved peak shape of succinic acid, but also for ion suppression that resulted in reduced sensitivity for MMA. Precipitation with 0.5% formic acid in methanol greatly increased sensitivity compared to TCA when evaluating the same sample (Figure 3). The methanol-based supernatant was compatible with direct analysis providing a sample volume of 3 µL or less was injected in order to avoid solvent effects.

Figure 2: 40% Trichloroacetic Acid (w/v) Protein Precipitation.

PeakstR (min)Precursor IonProduct Ion
1.Succinic acid1.54117.373.1
Methylmalonic Acid in Human Plasma (Trichloroacetic Acid Preparation)
LC_CF0719

Figure 3: 0.5% Formic Acid in Methanol (v/v) Protein Precipitation.

PeakstR (min)Precursor IonProduct Ion
1.Succinic acid1.60117.373.1
2.Methylmalonic acid1.96117.373.1
Methylmalonic Acid in Human Plasma (Formic Acid Preparation)
LC_CF0720

Chromatographic Performance

A fast, 5-minute analysis of methylmalonic acid (Figure 4) was obtained from the direct injection of the supernatant. MMA was clearly resolved (USP resolution of 3.0) from the isobaric interference, succinic acid, which made peak identification and quantitation easy. While analytical columns containing superficially porous particles can offer a speed advantage for some applications, a fully porous particle Force C18 column was used here because the additional surface area provides the retention required to chromatographically separate MMA from succinic acid.

Figure 4: 50 ng/mL MMA in SeraFlx BIOMATRIX.

PeakstR (min)Precursor IonProduct Ion
1.Succinic acid1.69117.373.1
2.Methyl-D3-malonic acid2.02120.276.2
3.Methylmalonic acid2.03117.373.1
Methylmalonic Acid in SeraFlx BIOMATRIX on Force C18 by LC-MS/MS
LC_CF0717

Linearity

Using linear 1/x weighted regression, the method showed good linearity for MMA across a range from 10–500 ng/mL with r2 values of at least 0.999 in all three accuracy and precision experiments.

Accuracy and Precision

Precision and accuracy analyses were performed on three different days. The method accuracy was demonstrated to be within 6.00% of the nominal concentration for all QC levels. The %RSD was 5.19%–6.50% and 7.67% for intra- and interday, respectively, at the LLOQ. The %RSD was 0.866%–6.24% and 1.97%–4.99% for intra- and inter-run, respectively, across the low, mid, and high QC levels, indicating good method precision (Table II).

Table II: Interday Accuracy and Precision of SeraFlx BIOMATRIX QC Samples.

Analyte

QC LLOQ (10 ng/mL)

QC Low (20 ng/mL)

QC Mid (150 ng/mL)

QC High (400 ng/mL)

Avg. Conc. (ng/mL)

Avg. Accuracy (%)

Precision (%RSD)

Avg. Conc. (ng/mL)

Avg. Accuracy (%)

Precision (%RSD)

Avg. Conc. (ng/mL)

Avg. Accuracy (%)

Precision (%RSD)

Avg. Conc. (ng/mL)

Avg. Accuracy (%)

Precision (%RSD)

MMA

9.40

9.40

7.67

19.0

95.1

4.99

152

101

4.84

404

101

1.97


Matrix Surrogate Evaluation

Because endogenous methylmalonic acid was found in double charcoal stripped human plasma (Figure 5), an evaluation of the use of a surrogate matrix for preparing calibration curves and QC samples was performed. SeraFlx BIOMATRIX has physical and chemical characteristics similar to human serum/plasma and tested negative for the presence of MMA. A calibration curve prepared in SeraFlx BIOMATRIX was used to quantitate the endogenous level of MMA in the double charcoal stripped human plasma. A concentration of 13 ng/mL was determined. The plasma was then spiked with additional MMA at concentrations corresponding to the LLOQ and low, mid, and high QC levels. The method accuracy was demonstrated to be within 7.00% of the nominal concentration for all QC levels. The method precision was 6.77%, which is within the acceptance criteria, for all QC levels, indicating that SeraFlx BIOMATRIX was an appropriate matrix surrogate for the analysis of MMA in human plasma (Table III). The suitability of this surrogate matrix for human plasma can improve lab efficiency by eliminating the need to prescreen control material for detectable levels of MMA.

Figure 5: Endogenous MMA (13 ng/mL) in Double Charcoal Stripped Plasma.

PeakstR (min)Precursor IonProduct Ion
1.Succinic acid1.68117.373.1
2.Methyl-D3-malonic acid2.02120.276.2
3.Methylmalonic acid2.03117.373.1
Methylmalonic Acid in Human Plasma on Force C18 by LC-MS/MS
LC_CF0718

Table III: Inter-Day Accuracy and Precision of Human Plasma QC Samples.

Analyte

QC LLOQ (23 ng/mL)

QC Low (33 ng/mL)

QC Mid (163 ng/mL)

QC High (413 ng/mL)

Avg. Conc. (ng/mL)

Avg. Accuracy (%)

Precision (%RSD)

Avg. Conc. (ng/mL)

Avg. Accuracy (%)

Precision (%RSD)

Avg. Conc. (ng/mL)

Avg. Accuracy (%)

Precision (%RSD)

Avg. Conc. (ng/mL)

Avg. Accuracy (%)

Precision (%RSD)

MMA

24.0

104

4.16

34.2

104

6.77

175

107

1.53

421

102

1.34


Conclusion

It was demonstrated that a simple, cost-effective sample preparation method can be used as an alternative to the tedious methods traditionally used for the analysis of MMA. The direct analysis of supernatant reduces costs associated with training, consumables, and extraction time. A Force C18 analytical column is able to successfully separate the naturally occurring isomer of MMA, succinic acid, with good resolution and a fast cycle time of only 5 minutes. SeraFlx BIOMATRIX was found to be a suitable matrix surrogate for the analysis of human plasma, eliminating the need to prescreen control material. With a fast, simple sample preparation procedure and 5 minutes of chromatographic analysis time, the established method provides a high-throughput assay for the clinical diagnosis of vitamin B12 deficiency and methylmalonic acidemia.

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