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Measurement of Folates in Serum and Conventionally Prepared Whole Blood Lysates: Application of an Automated 96-Well Plate Isotope-Dilution Tandem Mass Spectrometry Method

¹ Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341

^aauthor for correspondence: fax 770-488-4139, e-mail CPfeiffer{at}cdc.gov

Folate nutriture is associated with neural tube defects (1), vascular diseases (2), certain forms of cancer (3), and cognitive function (4). To reduce the risk for neural tube defects, the US Food and Drug Administration mandated folate fortification of cereal grain products beginning in January 1998 (5). Serum folate and whole blood folate (WBF) are measured to determine folate status. Clinical methods to determine serum folate and WBF, such as the microbiologic assay and various immunoassays, measure only total folate (TF) and present variable results, especially for WBF (6)(7). Few methods have described the measurement of WBF by chromatography-based methods (8)(9)(10)(11)(12)(13)(14)(15). Gas chromatography/mass spectrometry used to analyze WBF after cleavage of folates to p-aminobenzoic acid showed greater sensitivity than previous chromatographic methods and used for the first time an isotope-labeled internal standard (IS). The method, however, requires a complex and lengthy multistep sample preparation including chemical derivatization (11)(13)(14)(15). We describe here the first automated 96-well plate stable-isotope-dilution liquid chromatography–tandem mass spectrometry (LC/MS/MS) method that measures intact folate monoglutamates in conventionally prepared whole blood (WB) lysates and serum.

To increase sample throughput and minimize the extent of manual sample preparation, we adapted our recently developed manual solid-phase extraction (SPE) LC/MS/MS method for 5-methyltetrahydrofolic acid (5CH₃THF), 5-formyltetrahydrofolic acid (5CHOTHF), and folic acid (FA) in serum (16) to an automated method using 96-well plates. In addition, we extended this method to measure additional folate forms in WB lysates.

The folate calibrators, sample extraction, and chromatographic and MS conditions were as described previously (16). 5,10-Methenyltetrahydrofolic acid hydrochloride salt (5,10CH=THF) and 10-formylfolic acid (10CHOFA) were from Merck Eprova AG. We quantitatively isolated folates from 275 µL of serum or WB lysate (100 µL of WB diluted with 1 mL of 10 g/L L-ascorbic acid, incubated at room temperature for 2 h, and frozen at –70 °C until analysis) through SPE using a 96-well plate system (VersaPlate; Varian) and 1-mL phenyl cartridges (100-mg BondElut; Varian). The automated SPE was performed on an eight-probe system (Gilson 215; Gilson Inc.) that can process 96 samples in 5 h. We detected and quantified the folates in stabilized serum and WB extracts by positive-ion electrospray ionization LC/MS/MS. We used multiple-reaction monitoring (MRM) transitions for the following analytes (IS): 5CH₃THF (¹³C₅-5CH₃THF), m/z 460 (465)m/z 313; 5CHOTHF (¹³C₅-5CHOTHF), m/z 474 (479)m/z 327; FA (¹³C₅-FA), m/z 442 (447)m/z 295; tetrahydrofolic acid (THF; ¹³C₅-THF), m/z 446 (451)m/z 299; 5,10CH=THF (¹³C₅-5CH₃THF was used as pseudo-IS), m/z 456m/z 412; and 10CHOFA (¹³C₅-FA was used as pseudo-IS), m/z 470m/z 295. On the basis of MRM transitions, we are able to distinguish 5CHOTHF (m/z 474m/z 327) from 10-formyltetrahydrofolic acid (10CHOTHF; m/z 474m/z 298); however, during LC/MS/MS in our acidic mobile phase, 10CHOTHF converted within minutes to 5,10CH=THF and trace amounts of 5CHOTHF, THF, and 10CHOFA. When we added 10CHOTHF to serum or WB lysate, we recovered the amount added mainly as 5,10CH=THF (85%) and small amounts of 5CHOTHF, THF, and 10CHOFA. Thus, our method is not capable of quantifying 10CHOTHF. We quantified the formylated folates as 5CHOTHF and 5,10CH=THF, which are stable under these conditions (1% interconversion between each other). The tandem MRM profiles for various folates in the WB low quality-control (QC) pool are shown in Fig. S1 of the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue12/.

As with manual SPE, aqueous calibration curves obtained with automated SPE produced excellent linearity in the range 0.22–220 nmol/L with correlation coefficients between 0.998 and 1.000. The variability (CV) of the slope for the six folates over 10 days was 4.5% for 5CH₃THF, 6.0% for 5CHOTHF, 9.7% for FA, 9.7% for THF, 8.5% for 5,10CH=THF, and 12% for 10CHOFA. Using a signal-to-noise ratio of 3, we found in WB lysates limits of detection of 2.5 and 0.7 nmol/L for THF and 5,10CH=THF, respectively (corresponding to 51 and 13 fmol on column). The limits of detection for 5CH₃THF, 5CHOTHF, and FA in WB lysates were identical to the values we reported previously for serum (16).

Between-run variability for three serum QC pools (Table 1 ) was comparable to or slightly lower than the variability for manual SPE (16). Within-run variability (CV) (n = 3) for the same serum pools was 0.6–1.9% for 5CH₃THF, 1.4–7.0% for 5CHOTHF, and 3.0–13% for FA. Between-run variability for three WB QC pools (Table 1 ) was comparable to that of serum. Within-run variability for the WB low pool (n = 4) was 2.0% for 5CH₃THF, 5.6% for 5CHOTHF, 9.0% for THF, 8.6% for 5,10CH=THF, and 11% for 10CHOFA.

To assess method recovery and extraction efficiency, we added each analyte at two concentrations and in three replicates each to a low and/or medium WB QC pool. When each IS was added before SPE, we found nearly complete recoveries [mean (SD) recovery, 92.3 (1.8)% for 5CH₃THF, 93.0 (1.0)% for 5CHOTHF, 90.0 (3.8)% for FA, 99.6 (12.3)% for 5,10CH=THF, 101.2 (19.7)% for THF, and 93.8 (8.9)% for 10CHOFA]. When each IS was added after SPE, the extraction efficiency was close to 80% [77.1 (3.4)% for 5CH₃THF, 80.4 (4.1)% for 5CHOTHF, 81.2 (5.8)% for FA, 72.0 (11.7)% for 5,10CH=THF, and 74.1 (8.2)% for 10CHOFA] with the exception of THF, which had a lower extraction efficiency [46.3 (7.7)%; Table 1 ]. Recoveries of folates added to serum were identical to those we reported previously for manual SPE (16).

We investigated whether carryover from one row to the next and degradation of folates during automated SPE pose a problem. An entire row of water blank samples followed a row of high-concentration calibrator mixture (109, 53, and 57 nmol/L 5CH₃THF, 5CHOTHF, and FA, respectively) or a row of WB lysates enriched with various folates (218, 106, and 114 nmol/L 5CH₃THF, 5CHOTHF, and FA, respectively). We found <0.1% carryover into the water blanks. Three sets of serum and WB QC pools were extracted over 5 h in rows 1, 6, and 12 of the 96-well plate. The variability for all three sets was within the typical method variability, and the only degradation that we observed was for THF. The IS, however, corrected for it.

We evaluated the effect of sample type (i.e., serum vs plasma with various anticoagulants) by analyzing subject-matched specimens from 26 in-house volunteers and using serum as the reference value for each individual. Internal review board approval was obtained for all specimen collection. Concentrations of TF and 5CH₃THF in dipotassium EDTA, sodium heparin, and acid citrate dextrose (ACD) plasma and serum from serum separator tube (SST) samples showed highly significant Pearson correlations (r = 0.98–1.00) with their serum reference values. Deming regressions, Bland–Altman tests, and paired t-tests revealed a proportional bias for TF in the ACD plasma samples [–15.7% (95% confidence interval, –19.6% to –11.8%); P <0.0001], attributable mostly to the volume of the ACD solution in the tube, which inevitably diluted the samples by 12%. EDTA-plasma samples showed a smaller proportional bias for TF [–3.8% (–7.4% to –0.2%); P = 0.009]. The closest agreement for TF was between serum and SST [–0.8% (–3.2% to 1.5%); P = 0.63]. Heparin plasma gave slightly but not significantly lower concentrations [–2.5% (–4.6% to 0.5%); P = 0.11].

We analyzed folate forms in 38 human WB lysates from residual aliquots prepared for a folate interlaboratory comparison study performed by the Centers for Disease Control and Prevention in 2000 (7). A fresh aliquot of each sample was analyzed in a randomized, blinded way over 3 days. The mean between-run variability was excellent (Table 1 ). The mean (SD) and median WBF concentrations were 359 (151) and 327 nmol/L, with a range of 163–815 nmol/L. Depending on the methylenetetrahydrofolate reductase (MTHFR) genotype, we found different folate forms (Fig. 1 ). In individuals with the wild-type genotype (C/C; n = 24), we found mainly 5CH₃THF [90 (1)%] and small amounts of 5CHOTHF [10 ( (1))%]. Individuals with the C/T genotype (n = 8) displayed in general the same folate pattern [90 ( (2))% 5CH₃THF and 10 (1)% 5CHOTHF]. In one individual with the C/T genotype, we also found THF (87% 5CH₃THF, 8% 5CHOTHF, and 5% THF). In individuals with the T/T genotype (n = 6), we found significant amounts of 5,10CH=THF [10 ( (9))%] and THF [26 (21)%] in addition to 5CH₃THF [57 (28)%] and 5CHOTHF [7 ( (2))%]. One individual with the T/T genotype had only 5CH₃THF (92%) and 5CHOTHF (8%). We found no 10CHOFA in any of the individuals studied.

View larger version (27K): [in this window] [in a new window]   Figure 1. Folate forms in WB analyzed by LC/MS/MS. C/C, wild-type MTHFR genotype; C/T, heterozygous mutant MTHFR genotype (C677T); T/T, homozygous mutant MTHFR genotype (C677T).

These findings correspond well with earlier reports (12)(17). Bagley and Selhub (12) found that 5CH₃THF polyglutamates were the only folates in the erythrocytes of individuals with the C/C genotype and that formylated folates (quantified as the sum of 5,10CH=THF and 5- or 10CHOTHF) made up, on average, 30% (range, 0–69%) of the TF in erythrocytes of individuals with the T/T genotype. The preliminary results for individuals with the C/T genotype (n = 5) indicated that erythrocytes contained only 5CH₃THF polyglutamates. No THF was detected in the erythrocytes of individuals with either genotype. The discrepancies in findings between our work and that of Bagley and Selhub (12), i.e., we found small amounts of formylated folates in individuals with the C/C genotype and significant amounts of THF in individuals with the T/T genotype, might be explained by the difference in folate extraction. Bagley and Selhub (12) extracted frozen erythrocytes at high pH to prevent enzymatic deconjugation of folate polyglutamates. We used WB lysates generated at low pH (4.0) to obtain folate monoglutamates by the action of plasma pteroylpoly--glutamate hydrolase. Although this is the generally recommended and clinically used procedure for WB folates, it is conceivable that folate metabolism takes place during the lysis process. As a result, some folate forms could be found in conventionally prepared WB lysates that might not be present in native erythrocytes. The THF we find is most likely the dissociation product of 5,10-methylenetetrahydrofolic acid losing the C-1 group.

We analyzed 100 paired serum and WB lysate samples to assess what proportion of folates found in WB originates from serum. In general, we found no FA in WB lysates with the exception of those from two individuals who had serum FA concentrations >25 nmol/L, which spilled over into the WB lysate. Serum did not have quantifiable amounts of THF or 5,10CH=THF. On average, 95% each of 5CH₃THF and 5CHOTHF in WB lysates originated from erythrocytes, whereas the rest originated from serum. All of the THF and 5,10CH=THF found in WB lysates originated from erythrocytes.

The presented method measures folate monoglutamates only. If deconjugation of polyglutamates is incomplete, the TF concentration will be underestimated. Our findings on folate extraction from WB and the comparability of results obtained with different methods will be presented in a subsequent report.

This is the first reported method for measuring intact folate monoglutamates in serum and conventionally prepared WB lysates by LC/MS/MS using automated high-throughput SPE. The method displays excellent sensitivity and satisfactory precision, accuracy, and stability of folates during the 5-h SPE procedure. The specificity of this method is a clear advantage when it comes to quantifying individual folate forms. It can be a disadvantage when measurement of TF is of interest because the only folate forms measured are those that one is aware of. However, this highly specific method has a high potential of becoming a candidate reference method and helping to establish a standard reference material, which ultimately is needed to standardize clinical assays. The method’s automation and high sample throughput make it attractive for future use in clinical laboratories when further studies of the method have been completed.

Acknowledgments

We thank Dr. Les McCoy for input and thoughtful discussions related to the mass spectrometry aspect of the method and Dr. Michael Rybak for coordinating the blood collection to evaluate the effect of sample type.

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This Article Extract Full Text (PDF) Data Supplement All Versions of this Article: clinchem.2004.036541v1 50/12/2378    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (15) Citing Articles via Google Scholar Google Scholar Articles by Fazili, Z. Articles by Pfeiffer, C. M. Search for Related Content PubMed PubMed Citation Articles by Fazili, Z. Articles by Pfeiffer, C. M. Related Collections Nutrition Drug Monitoring and Toxicology Endocrinology and Metabolism Automation and Analytical Techniques