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Multianalyte quantification of vitamin [B.sub.6] and [B.sub.2] species in the nanomolar range in human plasma by liquid chromatography--tandem mass spectrometry.

Vitamins [B.sub.6] and [B.sub.2] are involved in the metabolism of homocysteine (1), a risk factor for occlusive vascular disease, Alzheimer disease, and adverse pregnancy outcomes (2). Vitamin [B.sub.6] serves as cofactor for cystathionine [beta]-synthase and cystathionine [gamma]-lyase, which convert homocysteine to cystathionine and then to cysteine (1), whereas vitamin [B.sub.2] modifies the phenotypic expression of the commonly occurring methylenetetrahydrofolate reductase (MTHFR) 677C[right arrow]T polymorphism and, thus, plasma concentrations of total homocysteine (3, 4). The metabolic pathways involving vitamin [B.sub.6], vitamin [B.sub.2], and homocysteine are illustrated in Fig. S-1 of the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol51/issue7/.

Vitamin [B.sub.6] exists in 7 forms: pyridoxine (PN), [1] pyridoxine 5'-phosphate (PNP), pyridoxal (PL), pyridoxal 5'-phosphate (PLP), pyridoxamine (PM), pyridoxamine 5'-phosphate (PMP), and the catabolite, 4-pyridoxic acid (PA; Fig. 1). PLP is the metabolically active form and is involved in ~100 enzymatic reactions. At present, it has not been established which [B.sub.6] vitamers) should be used as indicator of [B.sub.6] status (5), but several indices have been suggested, including plasma PLP (6,7), the combinations plasma PLP and PL (8), or plasma PLP plus PA (5,9,10).

[FIGURE 1 OMITTED]

Riboflavin serves as a precursor for flavin mononucleotide (FMN) and FAD (11). These coenzymes are involved in numerous reduction-oxidation reactions and take part in the metabolism of other vitamins, e.g., vitamin [B.sub.6] and folate (Fig. S-1 of the online Data Supplement). Plasma riboflavin appears to be a better indicator of vitamin [B.sub.2] status than are plasma FMN and FAD (12).

Measurement of vitamins [B.sub.6] and [B.sub.2] has become important for the assessment of homocysteine status (2). Overt vitamin [B.sub.6] deficiency is regarded as uncommon (6), but marginal deficiency may be related to an increased risk of cardiovascular disease (13,14). Vitamin [B.sub.2] deficiency may be more common than previously recognized, particularly in developing countries (15-17), among the elderly, in children, and in women during pregnancy (11).

Several methods for the determination of various [B.sub.6] vitamers in human plasma or serum have been developed (9,18-29), but at present no rapid and simple method exists for the determination of all 7 forms of vitamin [B.sub.6]. Vitamin [B.sub.6] methods are usually based on HPLC with fluorescence detection, which requires derivatization of PLP and complete chromatographic separation of the analytes. This often gives relatively long analysis times of ~30 min or longer (18,19,23,25-27). In studies involving vitamin [B.sub.6] in plasma, usually only PLP was determined (20,30-34), but in some cases (5,10,29,35,36), several [B.sub.6] vitamers were measured.

Analytical techniques for the determination of vitamin [B.sub.2] concentrations include fluorometric (37), liquid chromatographic (38,39), and capillary electrophoretic methods (40). Most HPLC methods have been designed for the detection of high concentrations of vitamin [B.sub.2] in food, pharmaceutical preparations, and urine (38). Some methods have been used for measurement of [B.sub.2] in whole blood (41-44), serum (42,43,45), or plasma (39,40,46-48).

We here describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of 7 vitamin [B.sub.6] species and 3 vitamin [B.sub.2] species in 60 [micro]L of plasma. The assay is sufficiently sensitive to quantify nanomolar concentrations of these vitamers.

Materials and Methods

MATERIALS

PL hydrochloride (99%), PLP (98%), PN hydrochloride (>99%), PM dihydrochloride (98%), PA (>99%), riboflavin (>98%), FMN (sodium salt; ~95%), and FAD (disodium dehydrate salt; >95%) were purchased from Sigma-Aldrich. PMP (98%) and heptafluorobutyric acid (HFBA; 99.5%) were purchased from Fluka, and PNP was kindly supplied as a gift from C.J. Argoudelis, Department of Food Science and Human Nutrition, University of Illinois-Urbana Champaign (Urbana, IL). Acetic acid (99.8%) and trichloroacetic acid (TCA; 99.5%) were from Merck, and acetonitrile (99.8%) was from SDS. Three deuterated species were used as internal standards. [d.sub.3] PL (99%) and [d.sub.8] riboflavin (98.3%) were synthesized by Beta Chem (Leawood, KS), and [d.sub.2] PLP (95%) was kindly supplied as a gift by S.P. Coburn, Department of Chemistry, Indiana University-Purdue University (Fort Wayne, IN). Doubly deionized water was used.

All vitamer calibrators were checked (using this assay) for the presence of other vitamin [B.sub.6] or vitamin [B.sub.2] forms. None were detected except in FAD, which contained ~% FMN. Likewise, no nondeuterated vitamers were detected in the deuterated internal standards [d.sub.2] PLP, [d.sub.3] PL, and [d.sub.8] riboflavin.

For routine assay calibration, we used plasma pooled from 4 persons (not taking B-vitamin supplements). The vitamer concentrations (nmol/L), as determined by this assay, were <4 for PMP, 87 for PLP, <0.2 for PNP, 35 for PA, 15 for PL, <0.1 for PN, <0.4 for PM, 71 for FAD, 8.3 for FMN, and 9.7 for riboflavin. To a fraction of this plasma pool we added 20 nmol/L each of PL, PN, PM, PNP, and riboflavin; 50 nmol/L each of PMP, PA, FMN, and FAD; and 100 nmol/L PLP. The calibration samples were aliquoted and stored at -80[degrees]C. The calibrators with added vitamins were prepared freshly at regular intervals of 14 days over 2 years. A constant ratio between endogenous and added vitamers indicated stability of the endogenous vitamins during storage.

For the matrix effect experiments, we obtained plasma from 9 healthy persons. For the recovery and precision experiments, we used plasma from a healthy blood donor, obtained at the blood bank at Haukeland University Hospital. Endogenous vitamer concentrations (nmol/L) were determined by this assay to be 59 for PLP, 14 for PA, 11 for PL, 60 for FAD, 8.4 for FMN, and 14 for riboflavin. The other vitamers were not detected in this plasma.

SAMPLE COLLECTION AND PROCESSING

EDTA plasma was obtained by collecting blood into Vacutainer Tubes (Becton Dickinson); the final EDTA concentration was 4 mmol/L. The samples were immediately put on ice and centrifuged (at 20008 for 10 min at 4[degrees]C) within 60 min. The plasma was then processed further or stored at -80[degrees]C until use.

Proteins were precipitated by mixing 60 [micro]L of plasma with an equal volume of TCA in water (50 g/L) containing the deuterated internal standards (100 nmol/L each of [d.sub.2] PLP and [d.sub.3] PL and 200 nmol/L [d.sub.8] riboflavin). The precipitation step was carried out by a robotic workstation (Plato 7; RoSyst Anthos). The solution was mixed for 30 s by the robot, left for 60 min on ice, and finally centrifuged at 60008 for 12 min. The supernatant (65 [micro]L) was transferred to a new vial, which was placed in a cooled (4[degrees]C) autosampler. The samples were protected from light.

INSTRUMENTATION

A series 1100 HPLC system (Agilent Technologies) equipped with a thermostated autosampler and a degasser was used for solvent delivery and sample introduction. The HPLC system was coupled to an API 4000 triple-quadrupole tandem mass spectrometer (Applied Biosystems/MDS SCIEX) outfitted with an electrospray ion source. A column switcher from Valco (type VMHA) was used to divert the flow to waste during the first 2.0 min and the last 2.2 min of the run. Analyst (Ver. 1.3.1; Applied Biosystems/MDS SCIEX) was used for data acquisition and analysis.

LC-MS/MS

We injected 50 [micro]L of deproteinized plasma into an Agilent stable-bond [C.sub.8] reversed-phase column [150 x 4.6 (i.d.) mm; particle size, 3.5 [micro]m] equipped with an Agilent stable-bond [C.sub.8] guard column [12.5 x 4.6 (i.d.) mm; particle size, 5 [micro]m]. The guard and analytical columns were mounted in a thermostated column compartment set at 20[degrees]C. The flow rate was 1 mL/min.

The mobile phase consisted of 3 components, solution A (650 mmol/L acetic acid), solution B (100 mmol/L HFBA in A), and solution C (900 mL/L acetonitrile in water).

The column was developed with gradient elution according to the following timetable: 0-0.2 min (97.5% A and 2.5% B), 6 min (28% A, 2.5% B, and 69.5% C), 6.1-6.6 min (2.5% B and 97.5% C), 6.7-8 min (97.5% A and 2.5% B). All gradient steps were linear.

Samples were injected every 8.75 min. The column effluent was delivered to the mass spectrometer with no split.

Acquisition settings were optimized by infusion of a 10 [micro]mol/L solution of each vitamer at a rate of 1 [micro]L/min.

Before entering the mass spectrometer, this solution was mixed at a T-junction with mobile phase delivered at a rate of 1 mL/min and with a composition corresponding to the time of elution of the actual analyte. The optimized conditions were adjusted according to 3 scan segments to acquire a sufficient signal quality, which is related to the number of data points across each chromatographic peak. The settings are detailed in Table 1. Curtain gas (10 psig), collision gas (4 psig), ion source temperature (490[degrees]C), ion source gases 1 and 2 (10 psig), and activated interface heater were identical for all segments.

The mass spectrometer was operated in the positive-ion mode. The vitamers were detected in the multiple-reaction monitoring (MR1VI) mode with unit resolution at both Q1 and Q3. The ion transitions for all analytes are shown in Table 1, which lists the protonated molecular ion [[M+H].sup.+] and the dominant product ion. Table 1 also shows the division of the chromatographic cycle into 3 scan segments, each of which encompassed 2-8 ion pairs.

For quantification, we included [d.sub.2]-PLP, [d.sub.3]-PL, and [d.sub.8] riboflavin as internal standards. The responses of PMP, PLP, and PNP were calculated as the ratios between the signal intensity areas for the vitamers divided by the area for [d.sub.2] PLP. Likewise, [d.sub.3] PL was used for calculating the area ratios for PA, PL, PN, and PM, and [d.sub.8] riboflavin for the vitamin [B.sub.2] species.

ASSESSMENT OF MATRIX EFFECTS

We investigated the matrix effects (49), which can be observed as ion enhancement or ion suppression (50), in plasma by measuring the peak intensities of all [B.sub.6] and [B.sub.2] vitamers in 9 different lots of plasma enriched with 30 nmol/L each of PN, PNP, PL, PM, riboflavin, and FMN and 150 nmol/L each of PLP, PMP, PA, and FAD after the protein precipitation step. The increases in peak intensities of the vitamers were compared with the respective intensities measured in an aqueous calibrator solution to which the same concentrations of analytes had been added. The matrix effect was calculated as:

Matrix effect (%) =

Peak area (added) - peak area (endogenous)/Peak area (aqueous solution) x 100

LINEARITY AND LIMITS OF DETECTION

It was impossible to obtain plasma devoid of all [B.sub.6] and [B.sub.2] vitamers by dialysis, as reported previously for PLP (20). We therefore prepared solutions containing 40 g/L albumin in phosphate-buffered saline, to which all vitamers were added to obtain final concentrations in the range 0.1-400 nmol/L. The albumin-phosphate-buffered saline solutions were used to assess linear ranges and limits of detection (LOD).

The signal-to-noise (5/N) ratio for each ion pair was calculated as S/N = (peak height - baseline)/SD (baseline), using the S/N script supplied by Applied Biosystems (Analyst Ver. 1.3.1). LOD were determined from the linearity data as the lowest concentrations that gave peaks with S/N ratios of 5.

RECOVERY AND WITHIN- AND BETWEEN-DAY CVS

The plasma was divided into 3 portions, and to 2 of these portions we added medium (10 nmol/L each of PN, PNP, PL, PM, riboflavin, and FMN and 50 nmol/L each of PLP, PMP, PA, and FAD) and high (30 nmol/L each of PN, PNP, PL, PM, riboflavin, and FMN and 150 nmol/L each of PLP, PMP, PA, and FAD) concentrations of vitamers. We analyzed 24 replicates at each vitamin concentration (low, medium, and high) in 1 analytical run, and recovery was calculated according to the formula:

Recovery (%) = Measured concentration - endogenous concentration/Added concentration x 100

We calculated within-day precision of the assay from the data of the recovery experiments and determined the between-day precision by assaying the same samples on 22 different days over a period of 54 days.

PLASMA CONCENTRATIONS OF VITAMINS

EDTA-plasma was collected from 94 nonfasting healthy individuals (35% male) with a median (range) age of 57 (11-93) years. To study the effect of [B.sub.6] supplementation, we collected plasma from cardiovascular patients (78% male) with a median (range) age of 61 (41-76) years, who received PN (40 mg/day; n = 30) or placebo (n = 30) for 1 month.

Results and Discussion

We have developed a high-throughput LC-MS/MS method for the measurement of 7 vitamin [B.sub.6] and 3 vitamin [B.sub.2] species in 60 [micro]L of plasma. The sample processing and analyte extraction involved a simple protein precipitation step with an equal volume of TCA, which was carried out by a robotic workstation.

PROTEIN PRECIPITATION AND LIBERATION OF THE ANALYTES

The precipitating agent (51,52) and incubation time (53) both affect the efficiency of protein precipitation and analyte extraction. TCA is efficient in precipitating human plasma proteins (51,52). Circulating PLP is almost entirely protein bound (54), and incubation of the plasma/ TCA mixture for 5 min at 50[degrees]C has been recommended to liberate all PLP (9).

Storage of the plasma/TCA mixture for 10 min at room temperature, or for 60 min at 0[degrees]C, provided maximum yields of PLP, PL, and FAD but had no effect on the other vitamers. All analytes were stable during incubation. We used these experiments to establish the routine procedure for sample processing.

CHROMATOGRAPHY

The vitamers included in the assay span a wide range of chemical properties. The [B.sub.6] vitamers contain a pyridine ring, whereas all [B.sub.2] vitamers have a tricyclic isoalloxazine moiety (Fig. 1). These structural features confer a large range of polarities and variable chromatographic behaviors on the analytes. The highly polarized [B.sub.6] species containing phosphate groups were poorly retained by the column, whereas elution of the larger [B.sub.2] species required an acetonitrile gradient.

We used a high concentration of acetic acid (650 mmol/L) to lower the pH of the mobile phase without adding TFA or other strong acids, which may suppress ionization (55) and thus reduce sensitivity. The mobile phase also contained HFBA, which is a volatile perfluorinated carboxylic acid suitable for LC-MS/MS (56). HFBA served as a counterion and was essential for the retention of PMP on the [C.sub.8] column.

The elution profiles of plasma to which all vitamers had been added are shown in Fig. 2, and the retention times are listed in Table 1. In plasma from healthy persons not taking B-vitamin supplements, only PLP, PA, PL, FAD, FMN, and riboflavin were detected (Fig. 2).

MS AND ACQUISITION SETTINGS

The mass spectrometer was used in MRM mode to optimize selectivity and sensitivity. All analytes gave a strong signal from the singly protonated molecule [[M+H].sup.+], and we selected the most abundant product ions from the fragmentograms. The selected molecular transitions are listed in Table 1.

The parent-product ion pairs for the nonphosphorylated [B.sub.6] species suggest loss of 2 x [H.sub.2]O for PN and PA, loss of [H.sub.2]O for PL, and N[H.sub.3] plus [H.sub.2]O for PM (57). For the remaining [B.sub.6] vitamers, the fragmentograms suggest additional loss of HP[O.sub.3] for PNP and PLP and loss of [H.sub.2]O for PMP compared with the nonphosphorylated [B.sub.6] forms. The ion transitions for the [B.sub.2] vitamers can be explained by elimination of the ribityl side chain for riboflavin and elimination of [H.sub.3]P[O.sub.4] for FMN (58). The product ion for FAD has the molecular mass of protonated adenylic acid (58).

[FIGURE 2 OMITTED]

There was no cross-talk between ion pairs originating from different analytes or between analytes and the corresponding internal standards.

The tandem mass spectrometer allows for optimization of acquisition conditions for each analyte, and we used specific settings for each MRM transition (Table 1). The mass spectrometer also permits skipping between the various MRM channels in time steps of milliseconds. To improve sensitivity, we restricted the number of parallel MRM traces to 8 by dividing each chromatographic cycle into 3 scan segments. The optimized time-scheduled MRM conditions for analyzing 10 vitamers and 3 deuterated internal standards are summarized in Table 1.

MATRIX EFFECTS

The mean (SD) matrix effect for 9 different plasma lots was 32 (2)% for PMP, 114 (10)% for PLP, 16 (2)% for PNP, 106 (9)% for PA, 87 (8) for PL, 48 (9)% for PN, 81 (18)% for PM, 527 (45)% for FAD, 100 (14)% for FMN, and 121 (4)% for riboflavin. Thus, ion suppression was most pronounced for PMP and PNP, whereas substantial ion enhancement was observed for FAD. Notably, the variability, also referred to as "relative matrix effect" (49), was low (<10%) for most analytes.

Both ion suppression and ion enhancement occur in LC-MS/MS (49, 59-61). A substantial matrix effect may contribute to low assay precision and low reproducibility. However, adequate assay performance and analytical recovery may be obtained if the relative matrix effect is low, if the external calibrators are composed in the same matrix as the samples, and if the internal standards correct for the matrix effect. Stable-isotope internal standards should afford optimal correction because the matrix effect is expected to be the same for an analyte and its labeled analog.

INTERNAL STANDARDS

A method based on mass spectrometry allows quantification by isotope dilution with inclusion of deuterated internal standards. This is in principle the ideal strategy to correct for variability in most analytical steps from analyte extraction to detection (62). We obtained 3 deuterated internal standards, [d.sub.2] PLP, [d.sub.3] PL, and [d.sub.8]-riboflavin, for the present assay. PMP, PLP, and PNP were corrected against [d.sub.2] PLP, the nonphosphorylated [B.sub.6] forms against [d.sub.3] PL, and vitamin [B.sub.2] species against [d.sub.8]-riboflavin. However, we did not observe better recovery or precision for the vitamers (PLP, PL, and riboflavin) measured with authentic deuterated standards compared with those measured in the absence of such standards (Table S-1 in the online Data Supplement and Table 3).

PERFORMANCE OF THE METHOD

We assessed assay performance in terms of linearity, sensitivity, analytical recovery, and precision.

For estimation of the linear dynamic range, we constructed calibration curves in the range 0.1-400 nmol/L (Fig. 3). Correlation coefficients ([r.sup.2]) >0.991 were obtained by least-square regression analysis for all compounds (Table 2).

LOD (defined as S/N ratio = 5) were calculated for all vitamers from the linearity experiments, and the results are listed in Table 2. LOD varied from 0.1 nmol/L for PN to 4 nmol/L for PMP and FAD. The LOD values obtained were comparable to those of other [B.sub.6] (20, 25, 27, 29) and [B.sub.2] (39) assays.

We investigated the recovery and precision of the assay by adding all vitamers at 2 (medium and high) concentrations to plasma containing endogenous vitamer concentrations.

Recoveries were in the range 78%-163% (Table S-1 of the online Data Supplement). Recoveries in the range 61%-110% have been reported for other vitamin B6 (9,18, 29, 63) and [B.sub.2] assays (39, 40). Thus, recoveries that differ from 100% are a common finding. One possible explanation for this may be uneven distribution of the analytes between the protein pellet and the supernatant. This may affect the accuracy of the assay but has minor effects on precision as long as the variability is low.

The mean within-day CVs were <10% for PLP, PL, PA, and riboflavin; l0%-15% for PN, PM, FAD, and FMN; and 15%-20% for PMP and PNP (Table 3). The between-day CVs were in general somewhat higher than within-day CVs (Table 3).

CVs were highest for PNP and PMP (Table 3). These were the compounds with the highest ion suppression. Thus, the low precision could be related to the relative matrix effect, which was not totally corrected by the internal standard, [d.sub.2] PLP.

For published [B.sub.6] assays, there are often no data on assay imprecision, or the imprecision reported varies widely, from 0.6%-5.6% (29), 2%-6% (9, 20), and 4%-12% (27), to 7%-6% (18).

The precision of the vitamin [B.sub.2] measurements seems to be somewhat lower than that of our vitamin [B.sub.2] capillary electrophoresis assay (40), but better than the precision reported for an HPLC assay (47).

STABILITY

All [B.sub.6] vitamers except PNP have been reported to be photosensitive and unstable in aqueous solutions (64-67). In plasma, PLP is unstable at room temperature (68) but is stable at low temperatures (-30[degrees]C) (69). FAD degrades at high TCA concentrations (40).

[FIGURE 3 OMITTED]

The aqueous stock solutions of vitamers; aqueous solutions of [d.sub.2]-PLP, [d.sub.3]-PL, and [d.sub.8]- riboflavin, and the EDTA-plasma used as calibrators were aliquoted and stored at -80[degrees]C. Under these conditions, we observed no degradation of the vitamers over a period of 2 years.

For protein precipitation, we chose a final TCA concentration of 25 g/L because at higher concentrations, we observed loss of FAD and a concurrent increase in FMN. After protein precipitation, the pH of the sample was ~1. We observed no instability for any [B.sub.6] or [B.sub.2] species in this acidic supernatant kept in subdued light at 4[degrees]C for up to 96 h (data not shown). Thus, the [B.sub.6] and [B.sub.2] vitamers are stable under the storage and sample handling conditions used for this assay.

PLASMA CONCENTRATIONS OF VITAMIN [B.sub.6] AND [B.sub.2] SPECIES

We analyzed vitamins [B.sub.6] and [B.sub.2] in 94 healthy individuals (35% male) with a mean (range) age of 57 (11-93) years. Median vitamer concentrations (nmol/L) were 35.4 for PLP, 22.4 for PA, 16.9 for PL, 63.1 for FAD, 7.5 for FMN, and 10.3 for riboflavin (Table 4). These concentrations are similar to those reported by others (9,22,27,40).

We also analyzed samples from patients with cardiovascular disease who received vitamin [B.sub.6] (40 mg/day PN; n = 30) or placebo (n = 30) for 1 month and observed that PLP, PL, and PA concentrations were 10- to 100-fold higher in vitamin [B.sub.6] supplemented patients than in the placebo group (Table 4). This difference was of a magnitude similar to that observed in previous studies of vitamin [B.sub.6] supplementation (36,63).

No patients in the placebo group, but some patients in the [B.sub.6] group, had detectable PN (n = 18) and PM (n = 5). PN and PM were detected only in 3 and 5 of the healthy individuals, respectively.

PNP was detected in most patients in the [B.sub.6] and placebo groups, but not in healthy persons, whereas PMP was not detected in any of the samples. Two other studies (28,63) have reported the presence of PNP in plasma. PMP has been found in plasma in some (19,25,26,28,70), but not all studies (18, 21, 22). It is uncertain whether the presence of PMP and PNP in plasma is the result of limited hemolysis.

The [B.sub.2] concentrations were similar in the [B.sub.6] supplemented and nonsupplemented groups.

In conclusion, the present method exploits the sensitivity and selectivity of the latest generation of tandem mass spectrometers for the measurement of all known forms of vitamins [B.sub.6] and [B.sub.2] in human plasma. The assay includes vitamers with a wide range of polarities, and the method has the advantage of high sample throughput and low sample volume requirement. The present method thus may be useful for large-scale studies of vitamins [B.sub.6] and [B.sub.2] in health and disease.

This work was funded by the Foundation to Promote Research into Functional Vitamin [B.sub.12] Deficiency. We thank S.P. Coburn for supplying us with deuterated PLP and C.J. Argoudelis for the PNP; we also thank Marit Krokeide and Anne-Kirstin Thoresen for excellent technical assistance.

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OIVIND MIDTTUN, * STEINAR HUSTAD, EINAR SOLHEIM, JORN SCHNEEDE, and PER M. UELAND

LOCUS for Homocysteine and Related Vitamins, and Section for Pharmacology, Institute of Medicine, University of Bergen, Bergen, Norway.

[1] Nonstandard abbreviations: PN, pyridoxine; PNP, pyridoxine 5'-phosphate; PL, pyridoxal; PLP, pyridoxal 5'-phosphate; PM, pyridoxamine; PMP, pyridoxamine 5'-phosphate; PA, 4-pyridoxic acid; FMN, flavin mononucleotide; LC-MS /MS, liquid chromatography-tandem mass spectrometry; HFBA, heptafluorobutyric acid; TCA, trichloroacetic acid; MRM, multiple-reaction monitoring; LOD, limits) of detection; and S/N, signal-to-noise.

* Address correspondence to this author at: LOCUS for Homocysteine and Related Vitamins, and Section for Pharmacology, Institute of Medicine, University of Bergen, Armauer Hansens Hus, N-5021 Bergen, Norway. Fax 47-55-97-46-05; e-mail nkbm@uib.no.

Received March 16, 2005; accepted April 12, 2005.

Previously published online at DOI: 10.1373/clinchem.2005.051169
Table 1. Retention times, molecular weights,
and optimized time-scheduled instrument settings.

 Transitions, m/z

 [t.sub.r], (a) Parent
 Analyte min [M.sub.r] ion Product ion

Scan segment 1 (0-3.5 min)
 PMP 2.4 248.2 249.3 232.1
 PLP 2.6 247.2 248.1 149.9
 d2-PLP 2.6 249.2 250.1 151.9
 PNP 2.9 249.2 250.2 134.1

Scan segment 2 (3.5-5.1 min)
 PA 4.3 183.2 184.1 148.0
 FAD 4.3 785.5 786.3 348.0
 PL 4.5 167.1 168.1 150.1
 d3-PL 4.5 170.1 171.1 153.1
 FMN 4.6 456.3 457.4 358.9/359.3 (b)
 PN 4.7 169.2 170.1 134.1
 PM 4.9 168.2 169.3 134.1

Scan segment 3 (5.1-8 min)
 RF 5.2 376.4 377.4 243.3
 [d.sub.s]-RF 5.2 384.4 385.4 251.1

 Analyte DT, ms IS, V DP, V CE, V CXP, V

Scan segment 1 (0-3.5 min)
 PMP 100 4500 41 19 6
 PLP 100 4500 90 24 12
 d2-PLP 100 4500 90 24 12
 PNP 100 4500 56 29 12

Scan segment 2 (3.5-5.1 min)
 PA 50 5500 61 31 12
 FAD 50 5500 106 31 12
 PL 100 4500 51 17 14
 d3-PL 100 4500 51 17 14
 FMN 100 5500 100 23 14
 PN 50 5500 66 29 10
 PM 100 4500 46 29 14

Scan segment 3 (5.1-8 min)
 RF 5.2 50 4500 90 30 6
 [d.sub.s]-RF 5.2 50 4500 90 30 6

(a) [t.sub.r], retention time; DT, dwell time; IS, ion spray; DP,
declustering potential; CE, collision energy; CXP, collision cell
exit potential; RF, riboflavin.

(b) For FMN, we used 2 product ions to increase the number of data
points over the peak.

Table 2. LOD and linearity.

 Regression parameters

Analyte LOD, nmol/L Slope Intercept, nmol/L [r.sup.2]

PMP 4.0 0.0069 0.00 0.999
PLP 0.4 0.015 -0.01 0.999
PNP 0.2 0.024 -0.08 0.997
PA 0.4 0.0058 0.02 0.999
PL 0.4 0.016 0.04 0.999
PN 0.1 0.012 -0.04 0.995
PM 0.4 0.011 -0.02 0.999
FAD 4.0 0.0025 -0.01 0.999
FMN 0.4 0.0039 0.04 0.991
RF (a) 0.2 0.0092 0.00 0.999

(a) RF, riboflavin.

Table 3. Precision of the assay. (a)

 Within-day CV, % (n = 24) Between-day CV, % (n = 22)

Analyte Low Medium High Low Medium High

PMP 20 18 18 19
PLP 5.7 6.1 5.1 7.4 5.5 6.4
PNP 19 17 22 19
PA 5.1 4.5 3.1 9.9 11 9.4
PL 9.2 3.4 3.5 8.5 7.6 6.4
PN 10 10 19 21
PM 11 11 19 17
FAD 15 10 8.3 11 11 11
FMN 18 12 13 22 13 15
RF 5.4 5.4 3.1 7.8 5.9 7.3

(a) The within-day experiments are identical to the recovery
experiments given in Table S-1 of the online Data Supplement.

Table 4. Vitamer concentrations in healthy persons
and cardiovascular patients. (a)

 Cardiovascular patients

 Healthy persons PN supplementation
Analyte (n = 94) Placebo (n = 30) (n = 30)

PMP ND (b) ND ND
PLP 34.4 (17.0-102.3) 30.8 (18.3-53.9) 356 (234-585)
PNP ND 2.6 (2.0-3.5) 2.5 (1.5-3.4)
PA 22.4 (11.2-88.0) 19.2 (12.6-26.6) 832 (262-2222)
PL 9.9 (5.7-28.2) 15.0 (9.6-20.1) 812 (170-2073)
PN ND ND (ND-0.1) 0.4 (ND-164)
PM ND 0.1 (ND-0.1) 0.3 (0.1-1.3)
FAD 63.1 (49.9-84.8) 75.0 (60.4-98.6) 75.2 (59.8-103)
FMN 7.5 (3.3-13.4) 7.0 (4.8-13.9) 6.9 (4.7-12.7)
RF 10.3 (4.9-38.4) 9.0 (5.6-21.8) 6.7 (3.2-24.4)

(a) Concentrations are given as the median (10th-90th percentiles).
All concentrations are in nmol/L.

(b) ND, not detectable; RF, riboflavin.
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Title Annotation:Automation and Analytical Techniques
Author:Midttun, Oivind; Hustad, Steinar; Solheim, Einar; Schneede, Jorn; Ueland, Per M.
Publication:Clinical Chemistry
Date:Jul 1, 2005
Words:7168
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