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Quantitative UPLC-MS/MS analysis of underivatised amino acids in body fluids
is a reliable tool for the diagnosis and follow-up of patients with inborn
errors of metabolism
W.A. Huub Waterval
, Jean L.J.M. Scheijen
, Marjon M.J.C. Ortmans-Ploemen,
Catharina D. Habets-van der Poel, Jörgen Bierau ⁎
Laboratory of Biochemical Genetics, Department of Clinical Genetics, Maastricht University Medical Centre, PO Box 5800, 6202 AZ, Maastricht, The Netherlands
a r t i c l e i n f o a b s t r a c t
Article history: Received 14 May 2009 Received in revised form 17 June 2009 Accepted 17 June 2009 Available online 24 June 2009
Keywords: Amino acids Tandem mass spectrometry LC-MS/MS Inborn errors of metabolism Validation Ninhydrin detection
Background: An electro-spray ionisation ultra-performance liquid chromatography tandem mass spectrometry
(UPLC-MS/MS) application for the quantitative analysis of amino acids was developed. The suitability for the
detection and follow-up of patients suffering from inborn errors of metabolism (IEM) was assessed by extensive
cross-validation with ion-exchange liquid chromatography (IEX-LC) with post-column ninhydrin derivatisation,
participation in external quality control (ERNDIM) and analysis of samples of patients with confirmed IEM.
Methods: Prior to analysis plasma and urine samples were merely diluted 150-fold in mobile phase. Amino acids
were detected in the multiple reaction monitoring mode (MRM) in the ESI-positive mode. The analytical results
were compared with IEX-LC. External quality control scheme performance is presented.
Results: Comprehensive analysis of amino acids in plasma and urine was achieved with a run-to-run time of
30 min. Validation results were satisfactory and there was a very good correlation between UPLC-MS/MS and IEX-
LC. Analytical results obtained in the external quality control scheme were essentially the same as those of the
other participants. Patients suffering from IEM were readily identified.
Conclusion: UPLC-MS/MS analysis of amino acids in body fluids is rapid, reliable and suitable for the diagnosis and
follow-up patients with IEM.
© 2009 Elsevier B.V. All rights reserved.
- Introduction
Quantitative analysis of amino acids (AA) in body fluids is the basis
for the diagnosis of inborn errors of metabolism affecting amino acid
metabolism. The most frequently applied method for the quantitative
analysis of physiological AA in body fluids is ion-exchange chromato-
graphy (IEX-LC) with post-column ninhydrin derivatisation [1]. This
technique is referred to as classic amino acid analysis and is performed
using dedicated equipment having excellent reproducibility. Moreover,
there is a vast world wide experience with this technique. Quantitative
analysis of all physiological amino acids is time-consuming, often
requiring 2 – 3 h per sample, and is not suited for high sample throughput. Moreover, numerous artefacts hamper the method and certain compounds cannot be separated using this technique. Tandem mass spectrometry (MS/MS) is a powerful technique for the quantitative analysis of small molecules. The technique is based on physico-chemical characteristics of analytes in an electrical field at near vacuum and subsequent collision induced fragmentation [2]. In general practice, a tandem mass spectrometer is operated in combination with high performance liquid chromatography (HPLC), which greatly increases its analytical performance [3,4]. Ions and other components from the sample matrix suppressing the signal output are removed and potential isobars are separated. HPLC-MS/MS facilitates rapid and specific analysis. Ultra-performance liquid chromatography (UPLC) has evolved from HPLC. Due to its greater analytical power, UPLC allows down scaling of sample volume and high throughput sample handling. UPLC-MS/MS supersedes HPLC-MS/MS in analytical performance [5,6]. In recent years the use of HPLC-MS/MS has become more popular in hospital laboratories and is applied to the quantitative analysis of small molecules such as metabolites and pharmaceuticals [2]. It is a powerful tool in the diagnosis of inborn errors of metabolism [7] and in newborn screening programs [8]. Most HPLC-MS/MS methods are dedicated applications for the detection or therapeutic follow-up for one specific disease or group of diseases. MS/MS applications covering
Clinica Chimica Acta 407 (2009) 36– 42
Abbreviations: AA, Amino acid(s); IEX-LC, Ion-exchange liquid chromatography; MS/MS, Tandem mass spectrometry; HPLC, High performance liquid chromatography; UPLC, Ultra-performance liquid chromatography; IS, Stable isotope-labelled internal standard(s); SSA, Sulphosalicylic acid; IEM, Inborn errors of metabolism; ERNDIM, European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism; TDHFA, Tridecafluoroheptanoic acid; MRM, Multiple reaction monitoring; LOD, Limit of detection; LOQ, Lower limit of quantification; ULQ, Upper limit of quantification; NTBC, 2-(2-Nitro-4-trifluoromethyl- benzoyl)-1,3-cyclohexanedione. ⁎ Corresponding author. Tel.: +31 43 387 78 35; fax: +31 43 387 79 01. E-mail address: jorgen.bierau@gen.unimaas.nl (J. Bierau). (^1) These authors contributed equally to the work presented.
0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.06.
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a wider range of disorders are the (semi-)quantitative analysis of
acylcarnitines for the detection of defects in fatty acid oxidation and
organic acidaemias [7,9] and the analysis of purines and pyrimidines
in urine [10,11]. It has been demonstrated in non-human samples that
analysis of underivatised AA by tandem mass spectrometry is possible
[12,13]. Piraud and co-workers demonstrated that HPLC-MS/MS
analysis of AA and related compounds for the detection of inborn
errors is in theory feasible [14–16]. Extensive data on the practicality
and validity of LC-MS/MS analysis of AA in comparison with the
established techniques have hitherto not been reported.
In our laboratory a project was initiated to replace the classic
amino acid analyser with a UPLC-MS/MS application. A reliable,
robust and simplified method was developed, validated and imple-
mented for the comprehensive quantitative analysis of underivatised
AA in body fluids using UPLC-MS/MS requiring minimal sample
volumes and manipulations. The analytical results were compared
with those of the classic amino acid analyser and of other laboratories
by means of participation in the ERNDIM external quality control
scheme. The suitability of the method to detect patients suffering from
inborn errors of metabolism is demonstrated.
- Materials and methods
2.1. Chemicals
AA standards and a prefabricated AA standard solution (A9906) were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Stable isotope-labelled (IS) AA were obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA). All solutions were prepared using LC-MS Ultra High Purity water and LC-MS-grade acetonitrile (J.T. Baker, Deventer, The Netherlands). Tridecafluoroheptanoic acid (TDFHA) 99% was obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). Sulphosalicylic acid (SSA) was obtained from Acros Organics (Geel, Belgium).
2.2. Patient samples
In 2008, all patient samples were routinely analysed using the method presented, in total 517 plasma samples and 365 urine samples were analysed. Samples of patients with known IEM were re-analysed. Urine samples from the patients with FIGLUria and prolidase deficiency were provided by Dr. L. Dorland, Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, The Netherlands. The urine samples from the patients with N-Aspartylglucosaminuria and Ornithine δ-amino- transferase deficiency were from the ERNDIM diagnostic proficiency testing scheme. Samples were used according to the “Code for proper use of human tissue” as formulated by the Dutch Federation of Medical Scientific Societies.
2.3. Standards
The purchased standard AA solution contained 500 μM of each AA. A stock solution containing 500 μM of L-S-Cys, PEA, L-Asn, L-Gln, L-Hci, DALA, Trp, L-Hcar, L-Sac, L-Aile, Asa was prepared in water. Prior to analysis this stock solution was mixed 1:1 (v/v%) with the purchased AA standard solution (A9906) resulting in final concentrations of 250 μM for each AA. A mixture of stable isotope-labelled AA was prepared in 0.1 M HCl and was used as internal standard for UPLC-MS/MS analysis. The concentration of the IS AA was between 70 and 800 μM. The stable isotope-labelled AA used are listed in Table 1.
2.4. Standard sample preparation for UPLC-MS/MS analysis
Patient samples were stored at −20°C and thawed in a water bath at 37 °C prior to analysis and homogenated by vortex mixing. Ten μl of plasma or urine was mixed with 10 μl of the internal standard solution and 1500 μl of 0.5 mM TDHFA in water. The sample was then ready for analysis.
2.5. Ion-exchange liquid chromatography (IEX-LC)
IEX-LC with post-column ninhydrin-derivatisation [1] was performed using a Biochrom-20 AA analyzer (Cambridge, UK) using the separation programme for physiological fluids supplied by the manufacturer. Patient samples were stored at −20 °C and thawed in a water bath at 37 °C prior to analysis and homogenated by vortex mixing. To 150 μl of plasma or urine 150 μl of 5% (m/v) SSA containing 300 μM aminoethylcysteine as the internal standard was added. The mixture was mixed thoroughly and incubated for 15 min on ice and subsequently centrifuged for 10 min at 25,000 ×g at 4 °C. The supernatant was then filtered through a 0.2 μm nylon filter (Costar, Corning, NY) for 1 min at 25,000 ×g at 4 °C. 170 μl of the filtrate was transferred to a clean injection vial. The injected volume was 50 μl.
2.6. Ultra-performance liquid chromatography conditions
Liquid chromatography was performed at 30 °C using a Acquity UPLC BEH C18, 1.7 μm, 2.1 × 100 mm column (Waters, Milford MA) and a gradient system with the mobile phase consisting of buffer A; 0.5 mM TDFHA and buffer B; 0.5 mM TFHA in acetonitrile (100%) at a flow rate of 650 μl/min (split less). The gradient programme used was: initial 99.5% A and 0.5% B; linear gradient to 70% A and 30% B in 14 min; hold for 3.5 min, return to initial conditions in 1 min at a flow rate of 700 μl/min, followed by equilibration for 10 min. One minute prior to the next sample injection the flow was set to 650 μl/min. Run-to-run time was 30 min. The injected volume was 5 μl.
2.7. Tandem mass spectrometry conditions
Mass spectrometry experiments and optimisation of the method were performed using a Micromass Quattro Premier XE Tandem Mass Spectrometer (Waters, Milford, MA). The mass spectrometer was used in the multiple reaction monitoring mode (MRM) in the ESI-positive mode. The desolvatation temperature was 450 °C, and the source temperature was 130 °C. The capillary voltage was set at 0.5 kV and the cone voltage was set at 25 V. Nitrogen gas was used as desolvatation gas and as cone gas. Nitrogen gas was produced using a NM30L nitrogen generator (Peak Scientific, Renfrewshire, Scotland). The cone gas flow was 50 l/h and the desolvatation gas flow was 800 l/h. Optimal detection conditions were determined by constant infusion of standard solutions (50 μM) in solvent A using a split system. MRM and daughter-ion scans were performed using argon as the collision gas at a pressure of 3.8 × 10−^3 mbar and a flow of 0.2 ml/min. Apparatus settings for detection and internal standards used are shown in Table 2.
- Validation procedure
3.1. Linearity, accuracy and detection limits
Because of the number of analytes and complexity of the sample matrices, linearity and accuracy (recovery) were determined by spiking 3 independent plasma, and urine samples and water with AA in dissolved water. Between 0 and 1000 μmol/l per AA was added to the sample matrix. The recovery was deduced from the slope of the calibration curve and corrected for the recovery in water to minimise differences between the different standards used. The limit of detection (LOD) was determined in water at a signal-to-noise ratio of 3, lower limits of quantification (LOQ) were determined in plasma and urine at a signal-to-noise ratio of 5.
3.2. Precision (intra- and inter-assay variation)
The intra-assay variation was determined by 10 consecutive analyses of basal and spiked plasma and urine samples. In order to obtain a sample with high concentrations of AA that normally are present in trace amounts 80–150 μmol/l of these AA were added. This was also done in a
Table 1 Twenty isotope-labelled AA used as internal standards with the specific mass transitions (MRM), declustering potentials (DP in V), collision energies (CE in eV), dwell times (DT in ms) and retention times (RT in min).
Abbreviation Monitored transition
DP CE DT RT
S-Sulfo- DL -Cysteine-2,3,3-D 3 S-Cys⁎ 204.8 N 122.8 25 12 5 0. Taurine-D 4 Tau⁎ 129.7 N111.8 25 10 5 0. L-Aspartic acid-2,3,3-D 3 Asp⁎ 136.75 N74.9 19 14 5 0. L-Asparagine-^15 N 2 Asn⁎ 134.8 N 74.9 19 15 5 0. DL -Serine-2,3,3-D 3 Ser⁎ 108.7 N 63 18 9 5 0. L-Glutamine-2,3,3,4,4-D 5 Gln⁎ 151.9 N 88 16 15 5 0. Glycine-2,2-D 2 Gly⁎ 77.7 N 77.7 20 3 5 1. DL -Glutamic acid-2,4,4-D 3 Glu⁎ 150.8 N 87 18 6 5 1. DL -Proline-2,3,3,4,4,5,5-D 7 Pro⁎ 122.7 N 77 22 14 5 1. DL -Cystine-3,3,3′,3′-D 4 (Cys) 2 ⁎^ 245.1 N122.1 20 20 5 1. DL -Alanine-2,3,3,3-D 4 Ala⁎ 93.7 N 93.7 17 3 5 1. DL -Valine-D 8 Val⁎ 125.7 N 80 17 12 5 4. L-Methionine-(methyl)-D 3 Met⁎ 152.7 N106.9 16 11 5 4. L-Tyrosine-ring-3,5-D 2 Tyr⁎ 183.8 N 137.9 19 17 5 5. L-Leucine-5,5,5-D 3 Leu⁎ 134.8 N 89 19 11 100 7. L-Phenylalanine-ring-D 5 Phe⁎ 170.8 N 124.9 20 15 5 8. DL-Homocystine-3,3,3′,3′,4,4,4′,4′-D 8 (Hcy) 2 ⁎^ 277.1 N140.1 16 10 5 8. L-Ornithine-5,5-D 2 Orn⁎ 134.9 N72.1 17 15 5 9. L-Lysine-4,4,5,5-D 4 Lys⁎ 151 N 88 22 20 5 9. L-Arginine-guanido-^15 N 2 Arg⁎ 177 N 70.2 22 20 5 9.
All standards were analysed in the ESI-positive mode.
was highly dependent on the concentration levels, low concentrations
exhibiting larger variations than high concentration levels. Satisfac-
tory results (CV ≤10%) were obtained for the majority of the AA in
spiked plasma and urine.
The between day variation observed in urine for PSer and PEA was
37% and 40%, respectively, reflecting the chromatographic challenges
for these AA. The CV's observed for Trp were 17% in plasma and 22% in
urine. Trp is labile in acidic conditions, probably causing the variation
observed for this AA. However, this would not hamper the diagnosis of
tryptophanuria, characterised by massively elevated concentrations of
Trp in plasma and urine [19]. This was tested by the addition of 100–
1900 μmol/l tryptophan to a plasma sample and a urine sample. The
samples were prepared for analysis as described and kept at 4 °C for
4 days. The recoveries were 99% for plasma and 94% for urine.
4.4. Correlation between the classic amino acid analyser and LC-MS/MS
The analytical results of the presented UPLC-MS/MS method were
compared with the results obtained using the Biochrom-20. Fifty
plasma samples were prepared for parallel analysis using both
methods. Sequential analyses introduced degradation artefacts caused
by freeze–thaw cycles and hampered comparison of the methods.
Spontaneous conversion of Gln to Glu, Asn to Asp and Arg to Orn
occurred in samples queued in the auto sampler of the amino acid
analyser and influenced the comparison. This was limited by reducing
the number of samples in each series. Because of the short run times,
this effect was negligible for the LC-MS/MS method. Linear correlation
curves were obtained for all AA physiologically present in plasma. For
most AA the slope of the curve was 1.0 ± 0.1, R 2 ≥ 0.9 and the mean
deviation between the two methods ≤10% (Table 3). Low concentra-
tions of some AA resulted in a lesser fit of the correlation curve as was
seen for EA and Abu. For Asp, Cit, and Trp the slope, R^2 and mean
deviation were outside the boundaries stated. The quantification of
Asp by IEX-LC is severely hampered by interference of a co-eluting
unidentified ninhydrin reactive compound, causing overestimation of
Asp. Moreover, Asp showed a highly variable elution time and peak-
form, dependent on the pH of the sample, causing erroneous peak identification. No base-line separation between Ala and Cit was achieved on the Biochrom-20 system, compromising the quantification of Cit, as Ala is present in far greater concentrations. The quantification of Trp using the IEX-LC method is hampered by two phenomena. First, Trp is subject to hydrolysis in acidic conditions. Because of the use of sulphosalicylic acid in the sample preparation procedure and the consecutive long time under acidic conditions in the auto sampler queue, the recovery of Trp is typically low. Second, no base-line separation was achieved between Trp and the flanking peaks of His and 3Mhis. The lack of base-line separation between Trp and His is also reflected in the poor correlation for His between the two methods. Furthermore there was a blank contribution for His in the LC-MS/MS method. In the case of Lys, an unidentified ninhydrin reactive compound eluted before Lys in some plasma samples. There was also a blank contribution for Lys in the LC-MS/MS method. The slope in the correlation curve was less than 0.9 for Hyp and Ser. Bland–Altman analysis showed that difference between the two methods is negligible for both compounds (Table 3). Also for Met the difference is extremely small. Thirty-five urine samples were prepared for parallel analysis using both methods. Comparison of the two methods in urine was hampered by the complexity of this matrix. The IEX-LC method suffered from many artefacts. Therefore no comparison was made for Cit, Hyp, Met, Pip, Lys, Asp, Pro, Aad, Abu, Leu, Trp, Hyl, Ans and Car. Concentrations were too low for S-Cys, PSer, PEA, Sar, (Cys) 2 , Sac, GABA, DALA, Aile, Asa, (Hcy) 2 , FIGLU and GlcNAc-Asn. For 11 out of 23 AA the slope was 1.0 ± 0.1, for the other AA the slopes were outside these limits (Table 4). R 2 was ≥ 0.9 and the mean deviation between the two methods ≤15% for the majority of the AA. For Thr, Hcy(ala), Hci, Ile and Phe the slope was less than 0.900, but the Bland–Altman analyses showed satisfactory similarity between the methods (Table 4).
4.5. Participation in the ERNDIM external quality control scheme
In 2008, we participated in the ERNDIM quantitative AA external quality control scheme using the UPLC-MS/MS method. The 2005– 2007 series were re-analysed. In the re-analysed samples reliable
Table 3 Comparison between UPLC-MS/MS and ion-exchange chromatography in plasma.
Amino acid
Slope Intercept R^2 Mean deviation (SD) (%)
Mean difference (SD) (μmol/l)
Mean (range) (μmol/l)
N
Tau 0.954 −4.631 0.962 10 (12) 9 (10) 85 (30–252) 46 Asp 0.740 −5.529 0.729 75 (37) 11 (5) 14 (6–49) 48 Hyp 0.855 3.188 0.902 −2 (24) 0 (5) 19 (5–67) 49 Asn 0.914 6.605 0.890 −6 (13) −3 (6) 46 (11–103) 48 Ser 0.896 12.44 0.917 −1 (8) −1 (9) 114 (72–191) 48 Gln 0.962 21.988 0.784 −1 (9) −5 (42) 445 (183–603) 48 Gly 0.952 5.346 0.949 3 (7) 7 (17) 251 (97–413) 47 Glu 0.855 4.587 0.965 11 (14) 12 (15) 108 (26–319) 47 Thr 0.949 −3.244 0.970 8 (7) 9 (8) 119 (44–235) 47 (Cys) 2 1.011 −0.776 0.964 7 (23) 1 (2) 9 (1–63) 46 Ala 0.976 −11.640 0.976 6 (6) 21 (23) 380 (96–805) 47 Cit 0.773 0.208 0.881 25 (14) 7(4) 28 (4–53) 46 Pro 0.902 9.645 0.954 5 (9) 11 (18) 205 (79–413) 47 Abu 0.949 −1.284 0.941 13 (8) 2 (1) 17 (6–31) 47 Val 0.975 −4.242 0.975 5 (5) 10 (10) 205 (43–330) 46 Met 0.905 −0.2346 0.962 11 (8) 3 (2) 23 (10–46) 46 Tyr 1.084 −4.869 0.953 −1 (8) 0 (5) 63 (33–115) 42 EA 0.661 2.116 0.748 11 (26) 1 (2) 9 (1–21) 47 Ile 0.906 0.288 0.947 10 (7) 6 (4) 61 (11–111) 47 Leu 0.986 −4.116 0.968 5 (6) 6 (8) 125 (43–252) 47 Phe 0.958 −4.041 0.948 9 (20) 5 (11) 58 (33–118) 43 His 0.665 13.577 0.708 19 (15) 15 (12) 77 (18–102) 48 Orn 0.901 3.063 0.977 6 (10) 5 (8) 77 (18–215) 47 Trp 0.727 20.784 0.460 −35 (35) −13 (13) 36 (3–74) 46 Lys 0.892 −10.613 0.942 19 (8) 29 (13) 157 (63–292) 47 Arg 0.954 −3.123 0.977 10 (7) 6 (5) 64 (9–146) 50
Correlation curves and Bland–Altman analysis are presented in the table.
Table 4 Comparison between UPLC-MS/MS and ion-exchange chromatography in urine.
Amino acid
Slope Intercept R^2 Mean deviation (SD) (%)
Mean difference (SD) (μmol/l)
Mean (range) (μmol/l)
N
Tau 1.059 7.424 0.987 −6 (11) −21 (34) 321 (12–1025) 35 Asn 0.942 6.491 0.972 −2 (12) −2 (10) 83 (12–219) 35 Ser 1.045 2.752 0.982 −5 (11) −13 (29) 269 (4–935) 35 Gln 0.993 14.233 0.996 −9 (24) −25 (71) 293 (8–815) 34 Gly 1.059 −22.767 0.983 −1 (6) −10 (38) 653 (233–1272) 20 Glu 0.975 2.582 0.977 −10 (14) −2 (3) 20 (3–87) 35 Thr 0.844 3.077 0.940 11 (20) 11 (20) 98 (20–296) 34 Ala 0.970 −3.069 0.992 −3 (6) −11 (18) 276 (48–789) 34 Aad 0.775 −1.976 0.928 −26 (25) −12 (12) 48 (3–159) 33 Hcy(Ala) 0.782 1.159 0.950 7 (20) 1 (2) 10 (2–23) 34 Hci 0.763 1.711 0.964 14 (33) 3 (6) 19 (1–93) 34 Val 0.956 −5.249 0.943 18 (15) 7 (6) 38 (13–87) 34 Tyr 0.956 1.183 0.983 3 (10) 2 (8) 58 (14–233) 33 EA 1.054 −5.267 0.988 −3 (9) −8 (22) 250 (65–238) 34 BAIB 1.022 −1.003 0.952 0 (18) 0 (10) 58 (9–184) 32 Ile 0.883 0.290 0.982 10 (12) 2 (2) 15 (4–36) 33 Phe 0.746 8.389 0.941 6 (20) 3 (9) 45 (6–116) 31 1-Mhis 0.968 10.123 0.906 11 (24) 27 (62) 257 (14–813) 29 His 1.323 −32.023 0.881 −23 (31) −118 (160) 524 (159–1272) 34 3-Mhis 1.352 − 31.704 0.986 −16 (26) −30 (49) 191 (24–254) 34 Orn 1.001 1.549 0.806 −12 (29) −2 (4) 13 (3–30) 34 Arg 0.604 10.992 0.427 − 33 (42) −5 (7) 17 (6–24) 33
Correlation curves and Bland–Altman analysis are presented in the table.
analysis of Gln, Glu, Arg, Orn and the sulphur-containing AA was not
possible. The ERNDIM scheme consists of 4 pairs of samples contain-
ing the various AA at 4 concentration levels. The inclusion of low
concentrations introduced very large standard deviations. The results
obtained with the UPLC-MS/MS method did not significantly deviate
from the results obtained by the other participants in the scheme
using mostly IEX-LC and reversed-phase HPLC analysis of derivatised
AA (Table 5). Exceptions were Aad and Abu in the 2005 series and Hyp
in the 2007 series. This may be caused by a matrix effect in the
samples or by the long storage of the samples. The separation of DALA
and Phe is difficult using IEX-LC, whereas this is no problem using LC-
MS/MS. This explains the higher recovery of DALA in comparison with
the other participants in the 2008 series. In the case of Hyl,
contribution of a co-eluting nin-hydrin reactive compound is often
seen in urine, hence the apparent lower recovery for Hyl observed for
our method.
In the 2008 series the concentration of arginine measured using
UPLC-MS/MS was structurally higher than the mean value of all
participants. As stated above, this is likely caused by a higher degree of
conversion of Arg to Orn in the classic AA analysis used by the majority
of the participants than in out method.
4.6. Patient samples
Plasma and urine samples of patients diagnosed with an inborn
error of metabolism were analysed. Table 6 shows that patients with
defects that lead to an accumulation of one AA or a group of AA were
readily identified. This is demonstrated for phenylketonuria (phenyl-
alanine hydroxylase deficiency), maple syrup urine disease (MSUD,
branched-chain α-ketoacid dehydrogenase deficiency), cystinuria,
argininosuccinate lyase deficiency, hypophosphatasia, aspartylgluco-
saminuria, FIGLUria, prolidase deficiency, ornithine δ-aminotransfer- ase defiency and ß-aminoisobutyric aciduria. The mentioned disorders are characterised by increased concen- trations of AA in the body fluids. 3-Phosphoglycerate dehydrogenase deficiency is a defect in the biosynthesis of serine. Patients present with modestly decreased Ser concentrations in body fluids. This is demonstrated in plasma (Table 6). The diagnosis of cystathionine ß-synthase (CBS) deficiency and molybdenum cofactor (MoCo) deficiency required additional ana- lyses. Whilst in CBS deficiency homocystine (Hcy) 2 , Met and Cys(hcy) accumulate, the concentration of homocysteine (Hcy) was deter- mined using a dedicated assay to establish the diagnosis. MoCo deficiency is characterised by a combined deficiency of sulphite oxidase and xanthine oxidase and therefore also required analysis of urinary purines and pyrimidines. In addition to the observed elevated excretion of S-Cys, the excretion of hypoxanthine and xanthine was strongly elevated while the excretion of urate was strongly decreased. In plasma a decreased concentration of Cys is observed. Furthermore, sulphite and thiosulphate can be detected in urine samples of patients with this condition. Therapy follow-up is shown for 4 patients. The patient with Tyrosinaemia type I (Fumarylacetoacetase deficiency) was on a Phe and Tyr restricted diet and was treated with NTBC, an inhibitor of 4- hydroxy-phenylpyruvate dioxygenase. In accordance with the ther- apy, a mildly elevated plasma concentration of Tyr and a sub-normal concentration of Phe were observed. Patients with isovaleric acidaemia have a deficiency of isovalery- CoA dehydrogenase, the second enzyme in the degradation pathway of Leu. Treatment consists of Leu (protein) restriction and supple- mentation with Gly and L -carnitine as detoxifying agents. Table 6 shows an acceptable low concentration of Leu and a modestly increased concentration of Gly. As a consequence of the diet, the concentrations of the Val en Ile were elevated. Ornithine transcarbamoylase (OTC) deficiency is an X-linked disorder, predominantly presenting in males, but females may be symptomatic as well. Table 6 shows normal concentrations of urea cycle intermediates in a female patient with OTC on a protein- restricted diet. In this sample the only aberrant AA was Gln, which was mildly elevated. The concentration of Gln together with the excretion of orotate is monitored to prevent hyperammonaemia. Lysinuric protein intolerance is a defect in the renal transport of the dibasic cationic AA Lys, Arg and Orn. Predominantly Lys is lost via the urine, resulting in a low plasma concentration as can be seen in Table 6. Due to a functional deficiency of urea cycle intermediates, hyperammonaemia occurs causing an elevated concentration of Gln in plasma. The patient was treated with Cit and protein restriction, which is reflected in the elevated concentration of Cit in plasma (Table 6).
- Discussion
A robust, rapid and reliable quantitative method for the analysis of AA in body fluids was developed, validated and implemented. This method has successfully replaced IEX-LC with ninhydrin derivatisa- tion as our standard protocol to analyse AA in body fluids. As demonstrated, the method is suitable to detect patients suffering from inborn errors of metabolism affecting AA metabolism. Moreover, this method could also be applied to non-IEM purposes, such as the measurement of sarcosine as a marker for prostate cancer [20]. The analytical performance of the total procedure is satisfactory and is superior to that of the classic AA analyser in the sense that the detection and quantification are specific, whereas with classic amino acid analyses the specificity is less. In general, the reproducibility is better in plasma than in urine. The major advantages of the method we present here are: 1) the minute volume of sample needed, 2) the simplicity of the sample preparation, 3) the short analysis time
Table 5 Comparison within ERNDIM quantitative amino acid scheme.
Amino acid
Measured values relative to median of all other participants (%) (SD) 2005 series 2006 series 2007 series 2008 series
S-Cys – X X 114 (25) Tau X X 91 (6) 87 (17) Asp X X X 99 (7) Hyp 115 (5) 115 (6) 128 (4) 118 (21) Asn X X X 97 (14) Ser X 117 (22) 159 (59) 95 (11) Sar 119 (22) 107 (16) 101 (32) 107 (12) Gln X X X 102 (11) Gly 93 (6) 94 (7) 109 (19) 101 (11) Glu X X X 103 (11) Thr 124 (15) 111 (36) 112 (15) 104 (9) (Cys) 2 X X X 89 (18) Ala 110 (10) 106 (4) 111 (7) 100 (9) Cit 87 (8) 91 (5) 100 (4) 108 (21) Pro 110 (11) 105 (6) 112 (14) 98 (5) Aad 124 (6) – – – Hcy(Ala) – 100 (7) 99 (6) – Abu 124 (6) 124 (11) 109 (11) 105 (11) Val 102 (9) 100 (5) 104 (10) 97 (6) Met X X 96 (10) 95 (5) Tyr 100 (3) 134 (88) 106 (9) 99 (6) DALA – – – 122 (30) Aile 109 (17) – – – Ile 103 (13) 107 (16) 114 (19) 112 (21) Leu 107 (12) 120 (26) 114 (25) 102 (5) Phe 108 (15) 124 (43) 116 (32) 99 (5) (Hcy) 2 – X X – 1-Mhis 97 (3) 98 (9) 101 (10) 105 (43) His 102 (9) 115 (15) 106 (5) 100 (8) Orn X X X 103 (16) Hyl – – – 86(7) Lys 105 (18) 114 (16) 102 (14) 95 (7) Arg X X X 131 (39)
X: Reliable measurement not possible, – : compound not included in the scheme.
identical to those of other participating laboratories. This is the first
report that documents both the qualitative and quantitative validity of
UPLC-MS/MS analysis of AA. It is clearly demonstrated that the
analytical performance of the UPLC-MS/MS method is at least equal to
other more widespread methods. The superior specificity greatly
enhanced the diagnostic power as artefacts hampering IEX-LC, such as
interfering ninhydrin reactive compounds, did not affect the method.
This was especially clear in the urine samples, in which the analysis of
AA by IEX-LC, and the consecutive interpretation, is not always
straightforward.
One must be aware that this type of instrumentation is highly
sensitive and multiple factors may influence the sensitivity and the
performance of the apparatus. This is demonstrated by the results
presented. However, this method has the major advantage that the
signal can easily be enhanced by analysis of less diluted samples.
The presented method was implemented in our laboratory in
January 2008 and has replaced analysis using the Biochrom-20 as the
routine method for the determination of AA in plasma and urine. This
action has greatly increased the promptness of our AA analyses and
has significantly enhanced the sample throughput capacity with the
same or even higher quality than previously.
Acknowledgements
The authors thank Marijn van Hulle and Peter Batjoens of Waters
Benelux for their technical support and advice. We thank Jean-Pierre
Bollen, Laboratory of Biochemical Genetics, Maastricht University
Medical Centre, for his technical support. We thank Dr. Bert Dorland
and Jaap Bakker M.Sc. for their advice. This work is dedicated to the
memory of Dr. Albert H. van Gennip.
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