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Articles |
1
Mayo Clinic, Department of Laboratory Medicine and Pathology, Biochemical Genetics Laboratory, 200 First St. SW, Rochester, MN 55905.
2
PE Biosystems, European Life Science Center, Langen
63225, Germany.
a Author for correspondence. Fax 507-266-4176; e-mail: rinaldo{at}mayo.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We developed liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) method based on the analysis of 100 µL of either plasma or urine with homocystine-d8 (2 nmol) added as internal standard. After sample reduction and deproteinization, the analysis was performed in the multiple reaction monitoring mode in which tHcy and Hcy-d4 were detected through the transition from the precursor to the product ion (m/z 136 to m/z 90 and m/z 140 to m/z 94, respectively). The retention time of tHcy and Hcy-d4 was 1.5 min in a 2.5-min analysis.
Results: Daily calibrations between 2.5 and 60 µmol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0.8 µmol/L, the signal-to-noise ratio for tHcy was 17:1. The regression equation for the comparison between our previous HPLC method (y) and the LC-MS/MS method (x) was y = 1.097x - 1.377 (r = 0.975; Sy|x =1.595 µmol/L; n = 367), and for comparison between a fluorescence polarization immunoassay (Abbott IMx; y) and LC-MS/MS (x) was y = 1.039x + 0.025 (r = 0.969; Sy|x =1.146 µmol/L; n = 367). Inter- and intraassay CVs were 2.9–5.9% and 3.6–5.3%, respectively, at mean concentrations of 3.9, 22.7, and 52.8 µmol/L. Mean recovery of tHcy was 94.2% (20 µmol/L) and 97.8% (50 µmol/L).
Conclusions: The sensitivity and specificity of tandem mass spectrometry are well suited to perform high-volume analysis of tHcy. Reagents are inexpensive and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The increased demand in clinical practice for measuring plasma tHcy raises the issue of developing new methods better suited to accommodate high testing volumes and faster turnaround time. Before the development of this method, tHcy was measured in our laboratory with an automated sample processor for sequential prepara- tion of plasma/serum and urine specimens. The methodology involves reduction of disulfides by sodium borohydride and dithioerythritol, derivatization of reduced sulfhydryls with monobromobimane, HPLC separation, and fluorometric detection (8)(9). This method is fully automated and relatively robust, but it requires the frequent analysis of quality-control samples to compensate for the lack of an internal standard and entails the use of five separate systems to meet the turnaround time needs of a high-volume clinical laboratory (150–200 samples/day). Although a fluorescence polarization immunoassay (FPIA) for plasma tHcy has been developed (10)(11) and is now widely used in clinical laboratories, we have sought the development of an alternative method for the determination of tHcy that takes advantage of the analytical versatility, specificity, and sensitivity unique to the combination of stable isotope dilution and electrospray ionization (ESI) tandem mass spectrometry (MS/MS) (12).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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sample preparation
A serum or plasma sample (100 µL) was mixed with 20 µL of
internal standard solution (2 nmol
homocystine-d8). When reduced,
homocystine-d8 yields
homocysteine-d4
(Hcy-d4) at double the initial
concentration. Complete reduction of disulfides was accomplished by the
addition of 20 µL of 500 mmol/L dithiothreitol, which was allowed to
react at room temperature for 15 min. Proteins were precipitated by the
addition of 200 µL of 1 mL/L formic acid and 0.5 mL/L
trifluoroacetic acid in acetonitrile. After 1 min centrifugation at
13 400g, 100 µL of the clear supernatant was
transferred to an autosampler vial. The preparation for urine differed
from that for plasma/serum only in the adjustment of urine pH to >7.0
with 1 mg/L NaOH before disulfide reduction and deproteinization.
Calibrators were prepared in plasma of known tHcy concentration
(typically <5 µmol/L) by the addition of a 200 µmol/L working
solution corresponding to tHcy concentrations of 0, 2.5, 5, 10, 20, 30,
40, 50, and 60 µmol/L. The endogenous concentration was derived from
the blank specimen and subtracted from the calibration points after
visual verification of results.
methods
A bench top triple quadrupole mass spectrometer API 2000
(Perkin-Elmer Sciex) operated in ion evaporation mode with the
TurboIonSpray ionization probe source (operated at 5800 V) was used.
Peripherals included a Perkin-Elmer Series 200 pump and an autosampler.
To enhance the stability of the signal, separation of tHcy and
Hcy-d4 from the bulk of the specimen
matrix was achieved by use of a short column (LC-CN, 3 cm x 4.6
mm; Supelco). Autosampler injections of 1 µL (corresponding to 0.3
µL of the original sample) were made using a mobile phase composed of
acetonitrile in 1 mL/L aqueous formic acid (600 mL/L acetonitrile-400
mL/L aqueous formic acid) at a flow rate of 1.0 mL/min. The column was
directly connected to the TurboIonSpray ionization probe operating with
the turbo gas on (6 L/min; sensor temperature, 250 °C) with the LC
column effluent flow-splitting set at 1:5. The retention times of tHcy
and Hcy-d4 were between 1.0 and 1.5
min in a 2.5-min chromatographic analysis. Total instrument acquisition
cycle time was 3 min per sample.
All results were generated in positive ion mode with the orifice voltage set at 36 V, automatically optimized using the protonated Hcy ion. For all MS/MS experiments, mass calibration and resolution adjustments (at 0.7 amu full width at half height) on both the resolving quadrupoles were automatically optimized using a poly(propylene)glycol 1 x 10-4 mol/L solution introduced via the built-in infusion pump on the API 2000.
Collisionally activated decomposition MS/MS was performed through the closed-design Q2 collision cell, operating with nitrogen at 0.06 kPa as collision gas. The 17 eV (lab frame) collision energy was adjusted automatically by the AutoTune algorithm.
MS/MS spectra were collected in continuous flow mode by connecting the built-in infusion pump directly to the TurboIonSpray probe. For MS/MS optimization, a 15 µmol/L Hcy solution was prepared in 500 mL/L acetonitrile-500 mL/L water containing 0.25 mL/L formic acid and infused at a flow rate of 10 µL/min. In the multiple reaction monitoring (MRM) mode, the instrument was optimized automatically by the built-in algorithm to monitor the 136.1 to 90 m/z and 140.1 to 94 m/z transitions for Hcy and Hcy-d4, respectively.
Data were acquired and processed using the MassChrom software (Ver. 1.1.1; Perkin-Elmer Sciex) including Multiview, Ver. 1.4, for chromatographic and spectral interpretation and Turboquan for Windows NT (Ver. 1.0; Perkin-Elmer Sciex) for the quantitative processing.
For the purpose of method comparison, an automated sample processor (model 232XL; Gilson) was used for sequential preparation and injection of plasma/serum and urine specimens. The methodology was adapted from previously published procedures (8)(9) involving reduction of disulfides using sodium borohydride and dithioerythritol, derivatization of reduced sulfhydryls with monobromobimane, HPLC separation, and fluorometric detection. Reversed-phase column chromatography was performed using a LC-18 column (15 cm x 4.5 mm; Supelco) and gradient elution with acetonitrile vs aqueous ammonium formate/ammonium nitrate. HPLC components included a Waters 600 solvent controller and Waters 474 scanning fluorescence detector (excitation, 365 nm; emission, 475 nm; 5-µL flow cell). Injections of 10 µL were made using the built-in LC switching valve on the sample processor. The total time to process and chromatograph a single specimen was ~40 min. Data were acquired and processed using Waters Millennium, Ver. 3.15, chromatography workstation software.
FPIA analysis for tHcy was accomplished using an IMx system from Abbott Diagnostics (10)(11). Instructions for use and analysis as well as reagents and supplies were obtained from the manufacturer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A. This spectrum was acquired by transmitting the protonated
molecular ion via Q1 and scanning the second
resolving quadrupole (Q3) for products resulting
from fragmentation in the collision cell. Loss of the carboxy moiety
from the protonated molecular ion of Hcy (m/z 136) yielded
the fragment at m/z 90. Using the autotune algorithm
provided in the system software, the instrument was optimized for
transmission of the protonated molecular ion and for maximum intensity
of the selected fragment. These results were used to design the MRM
experiment to sequentially transmit the m/z 136 protonated
molecular ion and the m/z 90 fragment via
Q1 and Q3, respectively.
Fig. 1B
shows an extracted MRM chromatogram obtained by analysis of a
mixture of tHcy (15 µmol/L) and
Hcy-d4 (40 µmol/L). The
retention time of tHcy/Hcy-d4 was
slightly influenced by mobile phase composition and pH (a lower pH
decreased the retention time).
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The extracted MRM chromatograms obtained from a specimen in which the
calculated tHcy concentration was 0.8 µmol/L is shown in Fig. 2
. The signal-to-noise ratio for the tHcy extracted MRM signal
was 17:1 (injected amount equal to 0.25 pmol); the ratio for the
Hcy-d4 (40 µmol/L) extracted MRM was
877:1.
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linearity
The interassay variability of calibration data obtained for
concentrations from 2.5 to 60 µmol/L on 6 consecutive days is shown
in Fig. 3
. The mean slope, intercept, and coefficient of linear
regression (r2) were 0.9925 (95%
confidence interval, 0.9697–1.0154), 0.0220 (95% confidence interval,
0.0110–0.0330), and 0.9971 (95% confidence interval, 0.9960–0.9982),
respectively.
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recovery, precision, and stability
The recovery and precision data are summarized in Table 1
. These experiments were conducted using as matrix bovine calf
serum diluted 1:1 with distilled water. Hcy (0, 10, or 25 µL of a 200
µmol/L solution) was added to 108 aliquots of bovine calf serum (100
µL), corresponding to final tHcy concentrations of 4, 24, and 54
µmol/L. Six aliquots of each set were prepared as described above and
analyzed in single determinations on each of 6 consecutive days. One of
each set of final samples was analyzed six times to verify the
instrumental precision of the method. Quantitative recovery and good
precision were obtained, demonstrating the accuracy of the present
method for the quantitative determination of tHcy. The stability of
prepared specimens was investigated by repeat injection of one set of 7
calibrators and 10 plasma samples (selected over a concentration range
of 5.5–36.1 µmol/L) with interim storage at 4 °C. The data
obtained are shown in Table 2
and indicate stable reduction of sulfhydryls and stability of
tHcy and Hcy-d4 stored up to 72 h
after sample preparation.
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method comparison
Leftover specimens of 367 analyses routinely performed by HPLC
over a 2-day period were reanalyzed on the same day by LC-MS/MS and
FPIA (IMx) in our laboratory (Table 3
). The correlation between the LC-MS/MS method and the HPLC
assay was y = 1.097x - 1.377. The
distribution of the mean vs the difference of paired tHcy values
[Bland-Altman plot, (13)] is shown in Fig. 4
. The correlation between the LC-MS/MS method and the FPIA assay
was y = 1.039x + 0.025; the corresponding
Bland-Altman plot is shown in Fig. 5
. These comparative data indicate equivalence of tHcy results
over the concentration range encountered in routine specimen analysis.
Because urine is not a suitable specimen for the immunoassay method, a
small group of urine specimens were analyzed by LC-MS/MS and HPLC
methods, and correlations similar to those presented for plasma were
obtained (n = 19; r2= 0.994;
y = 1.016x - 1.427).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Briefly, the principle of operation of an ESI-MS/MS system can be summarized as follows: ESI generates charged ions at near-atmospheric pressure from a solution nebulized as a fine spray of droplets by a high voltage electric field and/or pneumatically (19). Ion desorption from the droplets is induced by a counter flow of gas, and a fraction of the charged molecules is drawn in to the high vacuum of the quadrupole through a narrow opening. The first (Q1) or third (Q3) quadrupoles can be set either to scan a mass range or to select one or more individual ions. The second quadrupole (Q2) is used as a collision cell. When nitrogen is introduced into the Q2 region, fragmentation of ions passed or scanned through Q1 is enhanced by collisional activation, and the resulting fragment ions are then resolved by Q3. In the MRM mode, Q1 is set to transmit only the parent ions of interest (m/z 136 and m/z 140 for Hcy and Hcy-d4, respectively), Q3 scans only in the mass range of the daughter ions (m/z 90 and m/z 94) arising from the primary, collisionally activated fragmentation in Q2.
A variety of different procedures are currently available for the determination of tHcy (20)(21). Currently, HPLC-based procedures and a FPIA are the two methods most widely applied in clinical practice (11). Despite automation of sample preparation, our previous HPLC method (8)(9) required a visual verification of each profile by the operator and frequent manual corrections of baseline points at low concentrations. Inaccurate quantification because of interfering peaks or incomplete separation was a constant concern. The lack of internal standard and the relatively long instrument cycle time (17 min) were additional negatives. Although the FPIA is fully automated and faster than most HPLC methods, it could be affected by reagent batch variability and conceivably by other factors (22), and is not applicable to urine. Because urinary tHcy excretion has been reported to mirror plasma concentrations (2)(23)(24)(25), noninvasive determination of urinary tHcy could be regarded as the method of choice not only to monitor patients with homocystinuria but also as a tool to establish reliable pediatric reference ranges and to longitudinally study the impact of abnormal tHcy concentrations on cardiovascular disease from birth to adult life (26)(27).
Our results indicate that ESI-MS/MS provides a novel approach to the determination of tHcy in plasma and urine. Sample preparation is based on a simplified manual procedure (40 samples are prepared in <1 h including incubation time) that can be automated, requires inexpensive reagents and no derivatization, and takes advantage of a stable isotope-labeled internal standard with identical chromatographic behavior, which also serves as internal control of the reduction step. No interference from other compounds present in either serum/plasma or urine samples were noted in the analysis of >4500 samples.
The relatively high initial investment needed to acquire a tandem mass spectrometer has to be considered in the proper perspective: In our laboratory, for example, a single MS/MS instrument has replaced five sample processor/HPLC systems that were used to run 150–200 samples per day, with reductions of supply costs, space, personnel, and turnaround time of 35%, 80%, 29%, and 81%, respectively, whereas the equipment cost per sample increased only 25 cents per sample. The total (supplies plus equipment) cost per test has dropped 14%. Because the analysis of 200 samples requires no more than 10 h of instrument time, which could be conveniently set up overnight, one instrument can be very efficiently utilized for other applications or for development and research purposes.
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Acknowledgments |
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Footnotes |
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References |
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