The Analysis of Total Homocysteine in Plasma Using a Quattro Micro Tandem Mass Spectrometer

This application note describes the analysis of total homocysteine in plasma using a Quattro micro Tandem Mass Spectrometer.

Benefits

This method has been shown to be rapid, precise, and accurate for the measurement of total homocysteine

Total homocysteine (tHcy) is currently regarded as a risk factor for cardiovascular disease.¹ This increased interest has lead to a multitude of studies requiring the determination of total homocysteine in conjunction with other factors. There are various methods for measuring tHcy², including HPLC, enzyme immunoassay (EIA), GC-MS, and LC-MS/MS. A recent review³ suggests that GC-MS should be considered as the reference method. A comparison of GC-MS with other laboratory methods concluded that the methods were not interchangeable and reference material was urgently required.⁴ The most recent method for measuring homocysteine uses a deuterium-labelled internal standard and tandem mass spectrometry.⁵ This development requires no derivatization and therefore leads to an increase in sample throughput³ compared to existing techniques. We have developed an LC-MS/MS method that requires minimal sample volume, no centrifugation or transfers and therefore allows the possibility of automated sample preparation together with high throughput capability.

A Quattro micro Tandem Benchtop Mass Spectrometer fitted with a ZSpray ion source was used for all analyses. The instrument was operated in electrospray positive ionization mode and was coupled to a Waters Alliance HT HPLC System. All aspects of system operation and data acquisition were controlled using MassLynx NT v3.5 Software.

Maximum sensitivity and selectivity for homocysteine can be achieved by measuring product ions from the fragmentation of the protonated molecule [M+H]⁺. A solution of homocysteine (10 pmol/μL) was infused into the mass spectrometer and the cone voltage optimized to maximize the intensity of the [M+H]⁺ precursor (parent) ion (m/z 136). The collision energy was then adjusted to optimize the signal for the most abundant product ion (m/z 90), using argon as the collision gas at a pressure 5.0 x 10^-3 mbar. The product ion spectrum of homocysteine is shown in Figure 1. The process was repeated for the d₄-homocysteine analogue, which was used as an internal standard for quantification and to correct for losses during sample processing.

Plasma (10 μL) was aliquoted into a 96-well plate and d₈-homocystine, 10 μM, (10 μL) added and mixed for 1 minute. Dithiothreitol (DTT) 500 mM (20 μL) was added and mixed for 10 minutes at room temperature. 

Finally, water/0.1% formic acid/0.025% trifluoroacetic acid (100 μL) was added as a diluent and mixed for 1 minute. The resultant solution was then sampled (4 μL) directly from the microtitre well. 

The method uses an isocratic elution using a Waters Symmetry C₈ Column (2.1 x 100 mm, 3.5 μm) with aqueous 30% methanol/0.1% formic acid, at 250 μL/min. The cycle time (injection-to-injection) is ~2 minutes.

The MRM chromatograms for homocysteine and d₄-homocysteine are shown in Figure 2.

Plasma containing endogenous Hcy was spiked with a range of known Hcy concentrations (0 5, 10, 15, 25, and 50 μmol/L). The slope and positive intercept, calculated in QuanLynx Software, are used to calculate the endogenous value (7.96 μmol/L), as shown in Figure 3. The total homocysteine corrected for endogenous content is shown in Figure 4.

The performance characteristics of the assay were examined using two samples with homocysteine concentrations of 14.6 and 37.7 mM. Inter-assay results gave CV's of 5% and 8%, respectively (n=5) and the intra-assay (n=10) CV's for both samples was <2%. Validation of the method was performed using patient samples (n=50) kindly provided by Wythenshawe Hospital, Manchester, which were analyzed using both LC-MS/MS and an Abbot TDx Total Homocysteine kit. The LC-MS/MS method showed excellent correlation (r²= 0.9149) and slight positive bias (0.44) towards to LC-MS/MS method as shown in Figure 5.

This method has been shown to be rapid, precise, and accurate for the measurement of total homocysteine. 

The simplicity of the assay makes it ideal both for non-specialized staff and routine high throughput.

1. Boushey CJ., Beresford SA., Omenn GS,. Motulsky AG. A Quantitative Assessment of Plasma Homocysteine as a Risk Factor for Vascular Disease. Probable Benefits of Increasing Folic Acid Intakes. J Am. Med. Assoc. 274 (1995) 1049–1057.
2. Pfeiffer CM., Huff DL., Smith SJ., Miller DT., Gunter EW. Comparison of Plasma Total Homocysteine Measurements in 14 Laboratories: An International Study. Clin. Chem. 45 (1999) 1261–1268.
3. Rasmussen K., Moller J. Total Homocysteine Measurement in Clinical Practice. Ann. Clin. Biochem. 37 (2000) 627–648.
4. Ubbink JB., Delport R., Riezler R., Vermaak WJ. Comparison of Three Different Plasma Homocysteine Assays With Gas Chromatography-Mass Spectrometry. Clin. Chem. 45 (1999) 670–675.
5. Magera MJ., Lacey JM., Casetta B., Rinaldo P. Method for the Determination of Total Homocysteine in Plasma and Urine by Stable Isotope Dilution and Electrospray Tandem Mass Spectrometry. Clin. Chem. 45 (1999) 1517–1522.