Direct and simultaneous quantitation of 5-aminolaevulinic acid and porphobilinogen in human serum or plasma by hydrophilic interaction liquid chromatography-atmospheric pressure chemical ionization/tandem mass spectrometry

Article (PDF Available)inBiomedical Chromatography 27(2) · February 2013with142 Reads
DOI: 10.1002/bmc.2843 · Source: PubMed
Abstract
Serum/plasma concentrations of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre-treatment and derivatization steps. We report here a rapid, sensitive and specific hydrophilic interaction liquid chromatography-tandem mass spectrometry method for the direct and simultaneous quantitation of ALA and PBG in serum or plasma following simple protein precipitation with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode, following positive atmospheric pressure chemical ionization. Calibration was linear from 0.05 to 50 µmol/L for ALA and PBG. For both analytes, imprecision (relative standard deviation) was <13% and accuracy (percentage nominal concentrations) was between 92 and 107%. The method was successfully applied to the measurement of ALA and PBG in serum or plasma samples for the screening, biochemical diagnosis and treatment monitoring of patients with acute hepatic porphyrias. Copyright © 2012 John Wiley & Sons, Ltd.

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Available from: David C Rees, Jan 26, 2015
Direct and simultaneous determination of
5-aminolaevulinic acid and porphobilinogen
in urine by hydrophilic interaction liquid
chromatographyelectrospray ionisation/
tandem mass spectrometry
Christopher M. Benton
a,b
, Lewis Couchman
a
, Joanne T. Marsden
a
,
David C. Rees
c
, Caje Moniz
a
and Chang Kee Lim
a
*
ABSTRACT: Urinary concentrations of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with
acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre-treatment and derivati-
sation steps. We report here a rapid, sensitive and specic hydrophilic interaction liquid chromatographytandem mass spec-
trometry (LC-MS/MS) method, for the direct and simultaneous quantitation of ALA and PBG in urine following simple dilution
with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode,
following positive electrospray ionisation. Urine samples (N=46) from active and latent mutation-conrmed acute hepatic
porphyria patients and normal subjects (N=45) were analysed and the results compared with those of a commercially available
spectrophotometric method. The validated calibration range was 33000 mmol/L for ALA and 22000 mmol/L for PBG. For both
analytes, imprecision (relative standard deviation) was less than 5% and accuracy (percentage nominal concentrations) was
between 88 and 109%. The lower limit of quantitation was 0.1 mmol/L for both analytes. The calculated LC-MS/MS and spectro-
photometric results from patient samples compared well [Pearson correlation (r
2
) of 0.99 and 0.95, for ALA and PBG, respec-
tively]. The method was successfully applied to the measurement of ALA and PBG in urine samples for the screening, biochemical
diagnosis and treatment monitoring of patients with acute hepatic porphyrias. Copyright © 2012 John Wiley & Sons, Ltd.
Keywords: 5-aminolaevulinic acid; porphobilinogen; acute hepatic porphyrias; HILIC; electrospray ionisation tandem mass spectrometry
Introduction
5-Aminolaevulinic acid (ALA) and porphobilinogen (PBG; Fig. 1) are
precursors in the haem biosynthetic pathway. ALA-synthase rst
catalyses the condensation of glycine and succinyl coenzyme A
to produce ALA. Two ALA molecules subsequently condense
to form PBG, a reaction catalysed by ALA-dehydratase (ALA-D).
Hydroxymethylbilane-synthase (HMB-S) then catalyses the con-
densation of four PBG molecules to form the linear tetrapyrrole
hydroxymethylbilane, before enzymatic rearrangement and
cyclisation to form uroporphyrinogen III.
The analysis of ALA and PBG is important for the biochemical
diagnosis of the acute hepatic porphyrias, disorders caused by enzy-
matic defects in the haem biosynthetic pathway (Deacon and Elder,
2001; Floderus et al., 20 06; Pu y et al., 2010). They are particularly
relevant to the diagnosis of the most common acute porphyria,
acute intermittent porphyria (AIP), which is ca used specically by
reduced HMB-S activity. During acute attacks of AIP (obse rved
clinically as a number of neurovisceral symptoms), excessive produc-
tion and excretion of ALA and PBG results in substantially increased
concentrations of these metabolites being excreted in urine.
Unlike autosomal dominant acute hepatic porphyrias (AIP;
hereditary coproporphyria, HCP; variegate porphyria, VP), where
ALA and PBG concentrations are both raised during an acute
attack, increased concentrations of urinary ALA alone is a charac-
teristic feature of the very rare (autosomal recessive) acute hepatic
porphyria, ALA-D deciency porphyria (ADP) (Jaffe and Smith,
2007). Furthermore, ALA may be raised without PBG in the urine
of patients with lead toxicity and hereditary tyrosinaemia type I,
owing to the inhibition of ALA-D by lead and succinylacetone,
respectively. The simultaneous screening of ALA and PBG is
therefore crucial for the differential diagnoses of these conditions.
* Correspondence to: Chang Kee Lim, Clinical Biochemistry, KingsCollege
Hospital, Denmark Hill, London SE5 9RS, UK. E-mail: limchangkee@gmail.com
a
Clinical Biochemistry, Kings College Hospital, Denmark Hill, London SE5
9RS, UK
b
Cancer Studies and Molecular Medicine, RKCSB, University of Leicester,
Leicester LE2 7LX, UK
c
Department of Haematology, Kings College Hospital, Denmark Hill, London
SE5 9RS, UK
Abbreviations used: ADP, ALA-D deciency porphyria; AIP, acute intermit-
tent porphyria; ALA, 5-aminolaevulinic acid; ALA-D, ALA-dehydratase; ESI,
electrospray ionisation; HCP, hereditary coproporphyria; HMB-S, hydroxy-
methylbilane-synthase; PBG, porphobilinogen; SRM, selected reaction
monitoring; VP, variegate porphyria.
Biomed. Chromatogr. (2012) Copyright © 2012 John Wiley & Sons, Ltd.
Research article
Received: 26 April 2012, Accepted: 3 May 2012 Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.2770
Advertisement:
The most commonly used method for the clinical analysis of
ALA and PBG is based on the Mauzerall and Granick
method, which requires purication of urine samples using
separate anion- and cation-exchange columns (Bio-Rad, Hercules,
CA,USA),priortocomplexationwithEhrlichs reagent (para-
dimethylaminobenzaldehyde) and spectrophotometric detection
at 553 nm (Mauzerall and Granick, 1956). Whilst able to analyse
ALA and PBG in urine separately, the method is laborious,
nonspecic and prone to analytical interference from other
compounds, which react with Ehrlichs reagent (Marver et al.,
1966; Reio and Wetterberg, 1969; Pierach et al., 1977).
ALA and PBG are highly polar compounds and are not
sufciently retained using conventional reversed-phase chroma-
tography. Such methods are susceptible to signicant matrix
effects because of the insufcient retention of ALA and PBG
(Ford et al., 2001). A method using a nonvolatile ion-pairing
reagent (Crowne et al., 1981) has been reported, but such buffers
are incompatible with mass spectrometric operation. Other
liquid chromatographytandem mass spectrometry (LC-MS/MS)
methods have been investigated, but require lengthy and
convoluted sample preparation and derivatisation steps
(Floderus et al., 2006; Zhang et al., 2011), and the use of
expensive stable isotope-labelled internal standards. They are
therefore not suited to low-cost, high-throughput analysis in a
routine clinical chemistry laboratory.
Recently, hydrophilic interaction liquid chromatography
(HILIC) has become popular for the retention and separation of
polar compounds (Hemström and Irgum, 2006; Chauve et al.,
2010). Volatile buffers and mobile phases with high organic
solvent concentrations may be used. HILIC is therefore ideally
Figure 1. The chemical structures of 5-aminolaevulinic acid (ALA), porphobilinogen (PBG) and 6-amino-5-oxohexanoic acid (internal standard)
together with the likely sites of ionisation in positive [M + H]
+
and negative [M H]
ionisation mode.
C. M. Benton et al.
Biomed. Chromatogr. (2012)Copyright © 2012 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
suited to LC-MS/MS, since (i) desolvation/ionisation is signi-
cantly improved at the LC-MS interface; (ii) ion suppression
caused by matrix components that would be likely to co-elute
with polar compounds on reversed-phase systems are reduced;
(iii) evaporation steps during sample preparation are often
unnecessary; and (iv) LC column back-pressures are reduced.
Signicant increases in sensitivity (up to 8-fold) have been
previously reported for polar compounds analysed by HILIC in
conjunction with electrospray ionisation (ESI) MS/MS (Grumbach
et al., 2008).
We report here for the rst time, a rapid, sensitive and selec-
tive LC-MS/MS method for the simultaneous detection of ALA
and PBG in urine, without the need for extensive sample
preparation or derivatisation and expensive internal standards,
which will greatly facilitate the rapid, cost-effective clinical
analysis of acute porphyrias.
Materials and methods
Chemicals and reagents
ALA hydrochloride (98%), acetonitrile (LC grade), creatine,
4-hydroxyproline (99%), leucine (98%) and isoleucine (98%)
were purchased from Sigma Aldrich (Poole, UK). PBG (97%)
was purchased from Frontier Scientic Europe Ltd (Carnforth,
UK). Ammonium acetate (MS grade) was purchased from Fluka
(Poole, UK). Water was deionised (18.2 MΩ, Elga PureLab,
Marlow, UK). The internal standard, 6-amino-5-oxohexanoic acid
(Fig. 1) was synthesised (Gorchein, 1984) by and a generous gift
from Dr Abel Gorchein.
Apparatus
Microcentrifuge tubes (1.5 mL) were from Eppendorf (Cambridge,
UK). Samples, calibration solutions and internal quality control
(IQC) solutions were prepared using variable volume air-displacement
pipettes (BioHit, Finland), and acetonitrile was added using a repeat-
ing volume dispensing pipette (Multipette
W
, Hamilton). Extended
ne-tipped pastettes (Alpha Laboratories, UK) were used to transfer
prepared samples into 2 mL glass autosampler vials with
polypropylene snap-top caps (both Kinesis, UK). Other equipment
included vortex mixers (VWR, UK), 2 mL polypropylene screw-top
tubes (Alpha Laboratories, UK) and a microcentrifuge (Hermle Z233
MK-2). Weighings were performed using a ve-place analytical bal-
ance (Genius ME225D, Sartorius).
Patient samples
Residual human urine samples (N = 25) from active, symptom-
atic, mutation-conrmed AIP patients during or shortly after an
attack were analysed and the measured ALA and PBG concentra-
tions were compared with residual urine samples (N = 21) from
latent mutation-conrmed AIP patients and from normal
subjects (N = 45). Urinary creatinine was measured by the Jaffé
reaction using an automated clinical chemistry analyser (Advia
2400, Siemens).
Calibration and IQC solutions
Separate calibration and IQC stock solutions were prepared
containing ALA (6 mmol/L) and PBG (4 mmol/L) in pooled, pre-
tested urine, which had undetectable ALA and PBG
concentrations (<0.5 mmol/L for both analytes) using the Bio-
Rad method. Stock solutions were appropriately diluted with
the same pooled, pre-tested urine, to produce calibration stan-
dards and IQC solutions. Calibration standards (N = 6) were pre-
pared over the following concentration ranges: 33000 m mol/L
for ALA and 22000 mmol/L for PBG. IQC samples (N = 3) were
similarly prepared at concentrations of 70, 600 and 2000 mmol/
L for ALA and 20, 350 and 1000 mmol/L for PBG. After thorough
mixing and equilibration, calibration and IQC solutions were
stored in ca. 200 mL portions in polypropylene screw-top tubes
at 20
C until required. IQC solutions were run with each batch.
Clinchek
W
bi-level control materials from Recipe (Munich,
Germany), consisting of two samples prepared in lyophilised
urine, were used: level I (nominal ALA and PBG concentration
ranges 25.237.9 and 4.909.06 mmol/L, respectively) and level
II (nominal ALA and PBG concentration ranges 111.0166.0 and
49.582.2 mmol/L, respectively). Samples were reconstituted in
5.0 mL deionised water according to the manufacturers instruc-
tions and were stored in ca. 1 mL portions in 2 mL polypropylene
screw-top tubes at 20
C prior to analysis. Clinchek controls
were analysed with each batch.
LC-MS/MS sample preparation
Prior to LC-MS/MS analysis, samples, calibration and IQC solutions
(20 mL) were simply spiked with aqueous internal standard (10 mL,
100 mmol/L) and diluted with acetonitrile (170 mL) in a 1.5 mL
microcentrifuge tube. After vortex-mixing and centrifugation
(13,000 rpm, 2 min) to remove particulates, the supernatant was
transferred to an autosampler vial and capped prior to analysis.
LC-MS/MS parameters
The LC system was an Aria Transcend
TLX-II (ThermoFisher
Scientic Accela
600 pumps). The stainless steel LC column
(50 2.1 mm i.d) was packed with ThermoFisher Scientic Accu-
core
HILIC particles (2.6 mm average total particle diameter),
and protected by a guard column (10 2.1 mm i.d) packed with
the same material (ThermoFisher Scientic, Runcorn, UK). The
column oven temperature was maintained at 30
C (Thermo-
Fisher Scientic Hot Pocket
), the autosampler tray at 10
C and
the injection volume was 100 mL. Eluent A was 50 mmol/L aque-
ous ammonium acetate and eluent B was acetonitrile. Analytes
were eluted using a stepwise gradient elution programme (total
eluent ow-rate 1.0 mL/min). The elution programme was
started at 15% A (85% B) for 3 min; eluent A was then stepped
up to 18% (82% B) and ran isocratically from 3 to 6 min. Eluent
A was further increased to 50% A (50% B) and ran isocratically
from 6 to 9 min to clean the column, before returning to the
initial starting conditions to re-equilibrate the column (Fig. 2).
Eluent was split post-column in the ratio of 1:9 (100 mL/min
entering the MS) and was diverted to waste (i) for the rst
2.5 min of each analysis, and (ii) after elution of PBG, in order
to avoid impurities entering the MS/MS source during the LC
column aqueous wash step. Whilst eluent ow was running to
waste, the ESI capillary was washed with buffer-free acetonitrile
prior to the next injection (Fig. 2).
MS/MS (TSQ Vantage
, ThermoFisher Scientic, San Jose, CA,
USA) was carried out in both positive (quantitative) and negative
(qualitative) ionisation mode using ESI [spray voltage 3500 V;
capillary temperature 250
C; auxiliary, sheath and sweep gases
5, 45 and 0 (arbitrary units) respectively]. Data were collected
HILIC-MS/MS of ALA and PBG
Biomed. Chromatogr. (2012) Copyright © 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
in selected reaction monitoring (SRM) mode. The SRM transi-
tions for the precursor and product ions of ALA and PBG, to-
gether with analyte specic conditions, are displayed in Table 1.
Instrument control, and data acquisition and processing were
performed using Aria OS
(version 1.6.2), Xcalibur
(version
2.1.0, ThermoFisher Scientic) and LCQuan
(version 2.6.0,
ThermoFisher Scientic).
Statistical analysis
Two-tailed, paired t-tests were performed using GraphPad Prism
(version 5.04). Deming regression, BlandAltman plots and
Pearson correlations were calculated using Microsoft Excel
2003 and Analyse-it for Excel (version 2.21).
Method validation
The peak area ratios of analyte to internal standard were plotted
against analyte concentration to produce calibration curves, and
lines tted by least squares regression (quadratic, weighting
1/concentration
2
). Intra- and inter-assay precision (relative
standard deviation, RSD) and accuracy were measured by repli-
cate analysis (N = 6) of the IQC solutions on the same day, and
duplicate analyses (mean of duplicates) on different days
(N = 6), respectively. Loss of analyte during the sample dilution
step was investigated by comparison of the mean (N = 2) peak
area of each analyte from prepared solutions with the mean
(N = 2) peak area obtained from reference solutions at an equivalent
concentration. To investigate ion suppression/enhancement, human
urine samples (N= 10) with undetectable ALA and PBG
Figure 2. The LC gradient elution programme (% eluent B) is shown together with the divert valve positions (1 = to waste, 2 = to mass spectrometer).
Compounds isobaric with ALA (m/z 132) can be seen to elute when the LC eluent is diverted to waste. The peaks are leucine (0.55 min), isoleucine
(0.60 min), 4-hydroxyproline (1.15 min) and creatine (1.41 min). ALA, 6-amino-5-oxohexanoic acid and PBG were eluted at 4.01, 4.40 and 4.83 min, re-
spectively, during divert valve 2 when LC eluent was diverted into the mass spectrometer.
Table 1. The qualier ion (*) and quantier ion (**) selected reaction monitoring transitions for the precursor and product ions of
5-aminolaevulinic acid (ALA), porphobilinogen (PBG) and internal standard (IS), together with analyte specic conditions
Analyte Precursor ion (m/z) Product ion (m/z) Collision energy (V) S-Lens voltage (V) Polarity
ALA 132.1* 68.1 18 52 +
132.1** 86.1 12 52 +
130.1* 86.2 11 57
130.1* 112.2 11 57
IS 146.1* 110.1 8 56 +
146.1** 82.1 13 56 +
144.1* 59.1 14 49
144.1* 126.2 12 49
PBG 210.1* 122.1 23 67 +
210.1** 94.1 27 67 +
225.2* 152.2 15 75
225.2* 120.3 14 75
C. M. Benton et al.
Biomed. Chromatogr. (2012)Copyright © 2012 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
concentrations (<0.5 mmol/L for both analytes) using the Bio-Rad
method were analysed following addition of acetonitrile. The
detector response for each analyte transition was monitored
while an aqueous solution containing ALA (3 mmol/L) and PBG
(1 mmol/L) was infused (5 mL/min) via syringe post-column
(Bonglio et al., 1999). The lower limit of quantitation was ascer-
tained by replicate analysis (N = 5 at each diluted concentration)
of successive dilutions, using deionised water, of a urine standard
(1 mmol/L both analytes). The precision (% RSD) was calculated for
the replicates at each diluted concentration, and the limit of
quantitation taken as the lowest concentration where the % RSD
was less than 15 %.
Results and discussion
Assay performance/validation
The validated calibration range was 33000 mmol/L for ALA and 2
2000 mmol/L for PBG. Intra- and inter-assay precision and accuracy
data are summarised in Table 2. For both ALA and PBG, imprecision
(RSD) was less than 5% and accuracy (percentage nominal IQC con-
centrations) was between 88 and 109%. No loss of either analyte
was observed during sample preparation. The lower limit of quan-
titation was found to be 0.1 mmol/L for both analytes.
A typical mass chromatogram of a random urine sample from
an AIP patient taken during an acute attack is shown in Fig. 3. Rep-
resentative ion suppression chromatograms are also shown inset.
Interference from endogenous compounds known to be isobaric
with ALA (creatine, 4-hydroxyproline, leucine and isoleucine) was
excluded by analysis of individual solutions of these compounds.
No co-elution of these compounds was observed (Fig. 2).
Method comparison
The ALA and PBG concentrations (mmol/L) of patients with AIP
measured by LC-MS/MS compared well with those obtained
using the existing spectrophotometric (Bio-Rad) method.
Compared with the spectrophotometric method, the LC-MS/MS
method had a proportional bias of 0.89, constant bias of
17.19 mmol/L and r
2
of 0.99 for ALA and a proportional bias of
1.00, constant bias of 18.74 mmol/L and r
2
of 0.95 for PBG (Fig. 4).
However, the ALA and PBG concentrations in urine samples of nor-
mal subjects measured by LC-MS/MS were signicantly lower
(paired t-test, P < 0.0001 for both analytes) than those measured
by the Bio-Rad method (mean differences between methods
11.32 and 3.41 mmol/L for ALA and PBG, respectively, N =45).
The higher concentrations determined by the Bio-Rad method
Table 2. (a) Intra- and (b) inter-assay accuracy and imprecision data
IQC A IQC B IQC C
(a) Intra-assay (N = 6 at each concentration)
ALA Nominal (mmol/L) 70 600 2000
Mean measured (mmol/L) 70 586 1903
RSD (%) 2.5 1.0 3.1
Accuracy (% nominal) 99.7 97.7 95.2
PBG Nominal (mmol/L) 20 350 1000
Mean measured (mmol/L) 19 381 999
RSD (%) 4.7 1.6 4.2
Accuracy (% nominal) 97.2 109.0 99.9
(b) Inter-assay (N = 6 at each concentration)
ALA Nominal (mmol/L) 70 600 2000
Mean measured (mmol/L) 71 597 1951
RSD (%) 2.3 3.4 2.8
Accuracy (% nominal) 100.8 99.5 97.6
PBG Nominal (mmol/L) 20 350 1000
Mean measured (mmol/L) 18 343 971
RSD (%) 4.1 2.4 4.8
Accuracy (% nominal) 88.6 97.9 97.1
Figure 3. LC-MS/MS chromatogram of 5-aminolaevulinic acid (ALA,
m/z 132), 6-amino-5-oxohexanoic acid (internal standard, m/z 146) and
porphobilinogen (PBG, m/z 210) in positive [M + H]
+
ionisation mode,
from a patient with acute intermittent porphyria, during an acute attack.
The inset displays a tracing of the ion suppression, as described in the
text. ALA (1) and PBG (2) are represented by the black peaks.
HILIC-MS/MS of ALA and PBG
Biomed. Chromatogr. (2012) Copyright © 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
was probably due to interferences that also react with Ehrlichs
reagent, which is similar to previous ndings (Ford et al., 2001;
Floderus et al., 2006).
The use of BlandAltman plots showed a mean relative differ-
ence of 0.013 and 0.43 for ALA and PBG, respectively. The nega-
tive bias of PBG is due to the lower results calculated by LC-MS/MS
at low concentrations. The calibration and IQC urinary samples
used for LC-MS/MS analysis were also extracted and quantitated
using the Bio-Rad spectrophotometric method. The measured
ALA and PBG concentrations compared well between methods
except for the lowest standard (3 and 2 mmol/L for ALA and PBG,
respectively), where the Bio-Rad method measured 5-fold higher
for ALA and 2-fold higher for PBG.
LC-MS/MS
In the presented method, rapid polarity switching between
positive and negative ionisation modes allowed simultaneous
monitoring of ALA and PBG. This acted as an effective
conrmatory step, ensuring reliable results for each analysis.
Positive ionisation mode chromatograms were used to quanti-
tate ALA and PBG owing to an increased signal-to-noise ratio
compared with the equivalent negative ionisation data. In posi-
tive ionisation mode, the protonated [M + H]
+
PBG molecule
(m/z 227) showed signicant loss of NH
3
in the ESI source ([M +
H NH
3
]
+
) to give a stable, positively charged methylenepyrro-
lenine ion (m/z 210; Lord et al., 2000; Lim, 2009; Benton et al.,
2012), shown in Fig. 1. Owing to the increase in sensitivity that
MS/MS offers, only 20 mL of urine was required per sample for
analysis, in comparison to the 5001000 mL required by the spec-
trophotometry methods (Mauzerall and Granick, 1956).
HILIC provided retention and selectivity for these compounds.
The use of supercially porous particles (Accucore, ThermoFisher
Scientic) gave reproducible retention times and good ef-
ciency. Regions of ion suppression, as a result of simple dilution
as the only sample pre-treatment, were well-resolved from the
peaks of interest. The use of analytical guard cartridges served
to prolong column lifetime.
Figure 4. Method comparison of the LC-MS/MS assay and the Bio-Rad spectrophotometric method using latent and active AIP patient samples. (A)
Total ALA (mmol/L) measured with the Bio-Rad method compared with total ALA measure by LC-MS/MS (N = 45). The black dotted line is the line of
identity and the solid blue line is the Deming regression line [17.19 + 0.89x, r
2
(Pearson) = 0.99]. (B) Total PBG (mmol/L) measured with the Bio-Rad
method compared with total PBG measure by LC-MS/MS (N = 43) [18.74 + 1.00x, r
2
(Pearson) = 0.95]. Enlargements of the lower concentrations of
(C) ALA and (D) PBG are also shown. BlandAltman plots of (E) ALA and (F) PBG showing the mean concentration against the relative difference for
the Bio-Rad method vs LC-MS/MS. The mean difference with 2 SD of relative difference is shown (dashed lines).
C. M. Benton et al.
Biomed. Chromatogr. (2012)Copyright © 2012 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
Patient samples
ALA and PBG concentrations of normal subjects (N = 45) were
analysed by LC-MS/MS to establish a normal range. Concentrations
of ALA and PBG were <35 mmol/L (2.6 mmol/mmol creatinine) and
<1 mmol/L (<0.5 mmol/mmol creatinine), respect ively. Owing to
the specicity of the LC-MS/MS method, these are lower than the
current normal ranges established by sp ectroph otometry for
ALA and PBG, at <50 mmol/L (<3.8 mmol/mmol creatinine) and
<10 mmol/L (<1.5 mmol/mmol creatinine), respectively (Hindmarsh
et al., 1999; Deacon and Elder, 2001).
During an acute attack of AIP, the ALA and PBG concentra-
tions dramatically increased to 540 mmol/mmol creatinine and
2055 mmol/mmol creatinine, respectively (Figs 5 and 6). Intrave-
nous administration of haem is a common and effective treat-
ment for the acute porphyrias. The monitoring of such treatment
by LC-MS/MS is shown in Table 3. The lower PBG concentrations
seen in active patients (Fig. 6) are the measured concentrations
after haem treatment. During an attack of AIP, one sample had
an ALA concentration exceeding 3000 mmol/L and three samples
exceeded 2000 mmol/L of PBG.
The ALA and PBG concentrations were also measured in
latent AIP patients (N=21), some of whom had never had an
attack and others whose latest acute attack had been between
18 months and >10 years previously. Patients who had never
experienced an acute attack had ALA and PBG concentrations
similar to those of normal subjects. Interestingly, patients who
had previously had an acute attack still had very high ALA and
PBG concentrations even years after, much above the normal
baseline, suggesting that symptoms do not necessarily correlate
with ALA and PBG concentration (Gorchein and Webber, 1987).
Figures 5 and 6 show the difference in ALA and PBG concen-
trations (mmol/mmol creatinine) between normal subjects, latent
and active porphyria patients. The mean differences between
all the groups were statistically signicant (Tukeys multiple
comparison, one-way ANOVA, P < 0.05).
Conclusions
The presented LC-MS/MS method for the simultaneous quantita-
tion of ALA and PBG in urine samples is simple, rapid, sensitive,
specic and cost effective. The only sample preparation neces-
sary is dilution and centrifugation to remove particulates, thus
avoiding lengthy derivatisation and/or extraction procedures,
dramatically reducing manual preparation time. The assay
showed no loss of analyte during sample preparation, little ion
suppression at the retention times of interest, and known inter-
fering compounds were chromatographically resolved using
HILIC. This, together with specic SRM MS/MS transitions in both
positive and negative ionisation modes, will prevent false posi-
tives, as seen in current spectrophotometric methods, which
makes the method ideal for routine clinical use.
Figure 5. Box and whisker plot (median, interquartile range and
10th/90th perce ntiles) comparisons of ALA in normal subjects
(N =45), latent (N = 21) and active (N = 24) acute intermittent
porphyria patients using LC-MS/MS. Concentrations are expressed
as mmol/mmol creatinine and using a Log
10
scale. The mean differ-
ences between all the groups were statistically signicant (Tuke ys
multiple comparison, one-way ANO VA, p < 0.05).
Figure 6. Box and whisker plot (median, interquartile range and
10th/90th percentiles) comparisons of PBG in normal subjects
(N =45), latent (N = 21) and active (N = 22) acute intermittent
porphyria patients using LC-MS/MS. Concentrations are expressed
as mmol/mmol creatinine and using a Log
10
scale. The mean differ-
ences between all the groups were statistically signicant (Tuke ys
multiple comparison, one-way ANO VA, p < 0.05).
Table 3. Urine ALA and PBG concentrations (mmol/mmol
creatinine) in two acute intermittent porphyria (AIP) patients
during an acute porphyria attack and after haem therapy
Porphyria ALA
(mmol/mmol
creatinine)
PBG
(mmol/mmol
creatinine)
Clinical
details
AIP-1 14.2 31.2 During attack
AIP-1 10.5 25.0 1 day after haem
therapy
AIP-1 8.7 16.9 2 days after haem
therapy
AIP-2 3.7 28.1 During acute attack
AIP-2 2.8 6.3 3 days after haem
therapy
HILIC-MS/MS of ALA and PBG
Biomed. Chromatogr. (2012) Copyright © 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
Clinical application of this method has been demonstrated by
monitoring AIP patients on haem therapy during an acute porphyria
attack, together with the routine measurement of ALA and PBG con-
centrations in active and latent AIP patients. The results produced
compared well with the Bio-Rad spectrometric method.
Acknowledgement
We thank Dr Abel Gorchein for the generous gift of 6-amino-5-
oxohexanoic acid and Jeff Zonderman, Tom Whitehouse and
Sarah Robinson (ThermoFisher Scientic) for analytical support,
including the provision of instrumentation.
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    • "Hereditary coproporphyria increases the level of both ı-ALA and PBG, whereas the other diseases increase the level of ı-ALA only [23]. Although simultaneous determination of ı-ALA and PBG can be achieved using capillary electrophore- sis [6] and liquid chromatography–atmospheric pressure chemical ionization/tandem mass spectrometry [23], the current method is more sensitive than these methods and it can be used in laboratories equipped with GC–MS. "
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    • "The [oa]ToF pusher is then synchronised with the arrival of ion packets having the targeted m/z [20]. The assay described herein has incorporated hydrophilic interaction liquid chromatography (HILIC) which is particularly suitable for polar molecules [21]. An emphasis was put on having high throughput with an ambition of developing a rapid, reproducible method for clinical investigations. "
    [Show abstract] [Hide abstract] ABSTRACT: Objective: Acute heart failure (AHF) is associated with high mortality and morbidity. Trimethylamine N-oxide (TMAO), a gut-derived metabolite, has reported association with mortality risk in chronic HF but this association in AHF is still unknown. The present study investigated TMAO in patients admitted to hospital with AHF, and association of circulating levels with prognosis. Methods: In total, 972 plasma samples were analysed for TMAO concentration by liquid chromatography-mass spectrometry. Associations with in-hospital mortality (72 events), all-cause mortality (death, 268 events) and a composite of death or rehospitalisation due to HF (death/HF, 384 events) at 1 year were examined. Results: TMAO improved risk stratification for in-hospital mortality in combination with current clinical scorings (OR≥1.13, p≤0.014). TMAO tertile analyses reported a graded risk in adverse outcome within 1 year (OR≥1.61, p≤0.004) and improved outcome prediction when stratified as none, one or both biomarker(s) elevated in combination with N-terminal pro B-type natriuretic peptide (NT-proBNP) (OR≥2.15, p≤0.007). TMAO was independently predictive for death and death/HF when corrected for cardiac risk factors (HR≥1.16, p≤0.037); however, this ability was weakened when indices of renal function were included, possibly due to multicollinearity. Conclusions: TMAO contributed additional information on patient stratification for in-hospital mortality of AHF admissions using available clinical scores that include renal indices. Furthermore, elevated levels were associated with poor prognosis at 1 year and combination of TMAO and NT-proBNP provided additional prognostic information. TMAO was a univariate predictor of death and death/HF, and remained an independent predictor until adjusted for renal confounders.
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