349

Clinical Chemistry 42:3349-355 (1996) t4lecular PathoIo�y

Rapid diagnosis of homocystinuria and otherhypermethioninemias from newborns’ blood spots

by tandem mass spectrometryDONALD H. CIi�cE,I* STEVEN L. HILLMAN,1 DAVID S. MILLINGTON,1 STEPHEN G. KAHLER,1

BARBARA W. ADAM,2 and HARVEY L. LEVY3

We report a new method for the diagnosis of homocystin-

uria and other hypermethioninemias from dried blood spots

on newborn screening cards, based on isotope-dilution

tandem mass spectrometry. The mean concentration of

methionine in 909 unaffected newborns was 19 �mol/L

(CV 44%). The variability of results was reduced when the

concentration of methionine was expressed relative to that

of another amino acid in the same specimen. The mean

ratio of methionine to leucine plus isoleucine for these

same newborn blood spots was 0.16 (CV 25%). In newborn

samples from a collection categorized by a Guthrie bacterial

inhibition assay as true positive, unaffected, or falsely pos-

itive for hypermethioninemias, the ratio of methionine to

leucine for each true-positive specimen was at least 2.5

times greater than for respective age-matched unaffected

blood specimens. The ratio for falsely positive samples didnot differ from that for unaffected blood samples. We

predict that the ratio of methionine to leucine plus isoleu-

cine determined by tandem mass spectrometry will success-

fully detect hypermethiomnemias with very low rates for

false positives and false negatives.

��DEXIN� ThRMS: pediatric chemistry . heritable disorders

screening, metabolic

-ugh blood concentrations of methionine are found in two

nherited amino acid metabolic disorders: isolated hypermethi-

Mass Spectrometry Facility, Department of Pediatrics, Division of Biochem-cal Genetics, Duke University Medical Center, Box 14991, Research Triangle

ark, NC 27709.2 Newborn Screening Quality Assurance Laboratory, Division of Environ-

ental Health Laboratory Sciences, National Center for Environmental Health,enters for Disease Control and Prevention, Atlanta, GA 30341.

New England Regional Newborn Screening Program, State Laboratory

stirute, 305 South St., Boston, MA 02130.

* Author for correspondence. Fax 919-549-0709, e-mail dhchace@

pub.duke.edu.

Received September 21, 1995; accepted December 7, 1995.

oninemia and homocystinuria due to cystathionine 13-synthase

(EC 4.2.1.22) deficiency [1]. In isolated hypermethioninemia,

the degradation of methionine is reduced, at least in some cases,

by a deficiency of methionine adenosyltransferase (EC 2.5.1.6).

Individuals with isolated hypermethionmnemia have been normal

or, in a few instances, have had reduced cognitive function [2]. In

homocystinuria due to cystathionmne 13-synthase deficiency, the

hypermethionmnemia comes from overproduction of the homo-

cysteine that accumulates in persons with this disease. Primary

symptoms of this disorder include dislocation of the ocular lens

(ectopia lentis), mental retardation, neurological disturbances,

and skeletal defects. Presymptomatic diagnosis, usually through

newborn screening, followed by dietary restriction of methio-

nine and supplementation with cystine or simply treatment with

pyridoxine (vitamin B6) for those who are pyridoxine responsive,

has greatly improved the outcome of affected infants [1].

Newborn screening for both isolated hypermethioninemia

and homocystinuria is accomplished by analyzing methionine in

the dried blood spots collected routinely on filter paper. The

Guthrie bacterial inhibition assay is the semiquantitative

method used for this purpose [3]. Concentrations of methionine

>67 �.�mol/L (10 mg/L) are considered abnormal in some

laboratories, whereas other laboratories use a cutoff methionine

concentration of >134 p.mol/L (20 mg/L) [4]. With lower

protein intake because of increased breast feeding by neonates

[5] and with earlier newborn specimen collection because of

early hospital discharge [6], methionine concentrations in af-

fected neonates are likely to be only very slightly increased,

perhaps just above the 40 �mol/L (6 mg/L) concentration

considered the upper limit of normal for this amino acid in

plasma. Thus, false-negative results, already a recognized prob-

lem when newborn screening for homocystinuria is performed

by bacterial assay [6], will likely increase. Furthermore, the

number of false-positive results in the bacterial assay for methi-

onine is substantial.

We have developed a tandem mass spectrolnetric (MS-MS)

method for the analysis of methionine from dried blood spots in

a single test that also detects several other amino acids. Prey-

350 Chace et al.: MS-MS of homocystine in blood spots

ously, we validated this methodology for newborn screening of

phenylketonuria and tyrosinemia by measuring phenylalanine

and tyrosine, respectively /7], and for maple syrup urine disease

screening by measuring leucine/isoleucine and valine [8]. The

MS-MS method can now simultaneously measure methionine

and several other amino acids and acylcarniunes [9, 10] in one

test. Thus, the information for disease-specific quantitative

diagnostic components increasingly required for screening can

be efficiently obtained by the MS-MS method. Because of the

high selectivity and sensitivity of MS-MS, the frequency of

false-positive and false-negative results in measurements of

disease-specific metabolites is minimized. With further valida-

tion, the use of MS-MS should expand to allow for the detection

of many other disorders of newborns in a rapid and cost-

effective manner. Here, we describe our experiences with this

method for analyzing methionine in dried blood specimens from

newborns.

Matenals and Methods

SOLVENTS, REAGENTS, AND INTERNAL STANDARDS

High-purity methanol was obtained from Burdick and Jackson

(Muskegon, MI). Glycerol, sodium octyl sulfate, and methionine

were obtained from Sigma (St. Louis, MO). Butanolic-HCI

(3 mol/L) was obtained from Regis (Morton Grove, IL).

For internal standards we used stable isotopes obtained from

Cambridge Isotopes (Andover, MA); these included

‘3C� Iglycine, [2H4]alanine, [2H8]valine, [2H3jleucine,

[2H3]methionine, [2H5]phenylalanine, [2H4]tyrosine, and

[2H�]glutamate.

BLOOD SPECIMEN COLLECTION

The specimens tested consisted of the following: >16 000 driedblood spots from the North Carolina Division of Laboratory

Services Newborn Screening Program; a collection of blood

spots from newborns from the New England Regional Newborn

Screening Program (NERNSP); and a collection of blood spots

from the Newborn Screening Quality Assurance Laboratory,

National Center for Environmental Health, Centers for Disease

Control and Prevention (CDC), which was included as part of a

study on the extraction efficiency for methionine. The speci-

mens from the NERNSP had been previously analyzed by the

bacterial inhibition assay and stored at room temperature. They

were from infants categorized as unaffected, confirmed to have

either homocystinuria or isolated hypermethioninemia, or

falsely positive for hypermethioninemia. Each NERNSP spec-

imen from an affected infant was accompanied by two control

specimens matched for birth date and length of storage. The

specimens were sent to Duke University Medical Center for

blinded analysis. The specimen set from the CDC was prepared

by enriching specimens of whole blood with predetermined

concentrations of methionine and spotting them onto filter

paper. All specimens from the North Carolina Newborn

Screening Program, the NERNSP, and the CDC were collected

or prepared on S&S Grade 903 filter paper (Schleicher and

Schuell, Keene, NH).

SAMPLE PREPARATION

The semiautomated preparation of butyl ester derivatives of

amino acids from blood spots consists of a simple solvent

extraction and derivatization procedure that takes -2.5 h for a

batch of 60 samples. Two 3/16-in. (-4.8 mm)-diameter dots

were punched from a 1/2-in. (-12.7 mm)-diameter dried blood

spot into a 0.6-mL conical plastic vial. These two punched blood

dots are equivalent to 15.2 j�L of whole blood [11]. A methanol

stock solution of internal standards was prepared, containing 2.5

Mmol/L each of [2H4]alanine, [2H8]valine, [2H3]leucine,[2H1]methionine, [2H5}phenylalanine, [2H4}tyrosine, and

[2H3lglutamate and 12.5 p.moVL [‘5N1,’3C1]glycine. A Model

222 sample changer (Gilson Instruments, Middleton, ‘Wi) was

used to add 400 �L of this stock solution to each vial in the

60-sample rack. The sample rack was placed on an orbital shaker

for 30 mm. The sample changer then transferred the supernates

to corresponding 1 -mL flat-bottom vials in another rack, where

the samples were evaporated to dryness at 50 #{176}Cunder a gentle

stream of nitrogen with a custom-designed warm air incubator

(Grey Line Engineering, Churchton, MD). The sample changer

was then used to add 50 j�L of 3 moVL HCI in n-butanol to each

vial. The 60 vials contained in the rack were sealed with a bilayer

cover made from a sheet of septum material (bottom layer) and

a steel weight (top layer), placed in a forced-air oven, and

incubated at 65 #{176}Cfor 15 mm. After removal of the bilayer

cover, the vials containing the samples were placed in the warm

air incubator, and excess HCI-butanol was evaporated to dryness

under dry nitrogen. These derivatized samples were reconsti-

tuted with 35 �tL of an equivolume methanol:glycerol solution

containing sodium octyl sulfate, 1 g/L, and the vials were sealed

with Teflon-lined caps. The samples were then ready for

analysis by MS-MS.

To estimate the linearity of this assay, we enriched 200-�L

aliquots of whole blood from a single donor with 0, 2, 5, 10, 20,

50, and 100 nmol of added methionine. These samples were

spotted onto filter paper and dried overnight. To estimate

recovery (extraction efficiency) of methionine from blood spots,

we prepared two sets of four 1 .0-mL aliquots per set from a

single donor pool and enriched them with 0, 50, 100, and 200

nmol of added methionine. One set of samples was spotted onto

filter paper and dried overnight. These samples were then

prepared for analysis by the extraction and derivatizauon pro-

cedure described above. To the second set of samples we added

80 nmol of [2H3]methionine per aliquot and mixed well. These

whole-blood samples were spotted onto filter paper, drie

overnight, and extracted with pure methanol containing n

internal standards; the remainder of the sample preparatio

procedure was as described above.

Instrument variability was measured by performing 10 rep

licate injections of the singly derivatized product of one sample

Assay variability was measured by performing analyses of 1

control samples prepared from the same blood on one da

(intraday variability) and over a period of several weeks (interda

variability). At the CDC, a set of 11 methionine-enriched drieblood specimens was prepared from a single batch of whol

blood that had been frozen to lyse the erythrocytes. Methionin

enrichments of the specimens in this set ranged from 0 to 67

206

Product Ions of m/z 206

56 10461

41

101

5)a)t� 500

a)>a

c

101

CoCa)C

C 500

aII)

[-102)

40 60 60 100Mass

206

120 140/ Charge

i�0 180 200

Neutral Loss of 102

260 zeu suu

H

Clinical Chemistry 42, No. 3, 1996 351

�.tmol/L (0-100 mgfL blood). These samples were prepared as

described above.

MASS SPECTROMETRY

A VG Quattro triple-quadrupole tandem mass spectrometer

with Lab-base data system (Fisons Instruments, Danvers, MA)

was used and operated in the static liquid secondary ionization

mode. This mode incorporates an ion source containing a

cesium ion gun operating at 10 keV and a manually operated

insertion probe. Positively charged molecules are detected after

their separation in the first mass analyzer region (MSI) and in

the final mass analyzer region (MS2). An intermediate quadru-

pole located between the first and third quadrupole is used as the

collision region into which argon gas is introduced.

Tuning of the instrument is optimized by using a solution

containing deuterium-labeled standards prepared as butyl esters

as described previously [7, 8/. Product ion scans were produced

by focusing MS1 on the molecular mass (M + H)� of the butylester of methionine (m/z 206), whereas MS2 was used to scan

product ions between m/z 25 and m/z 215. The mass spectrum

obtained showed the fragmentation for each molecular ion of

methionine. Neutral loss scans of 102 Da were produced by

scanning MS1 from m/z 125 to m/z 300 while simultaneously

scanning MS2 at a mass range 102 Da lower, m/z 23 and m/z

198. This resulted in a spectrum of product ions corresponding

to (M + H - l02)�.

Quantification of methionine is achieved by converting the

ion abundance ratios of methionine:[2H3]methionine (m/z 206:

209) to the concentrations of methionine by reference to a

calibration curve. The calibration curve was generated from the

analyses of blood spots containing serially added known concen-

trations of methionine.

ResuttsANALYSIS OF METHIONINE BY MS-MS

The fragmentation pattern of the protonated molecular ion, [M

+ H1� (precursor ion, m/z 206), of methionine is shown in Fig.

1. A loss of the elements of butyl formate (102 Da) was

demonstrated by the presence of the product ion at mIx 104.

The precursor ion, mlz 209, for the internal standard,

[2H3]methionine, exhibited a similar fragmentation pattern with

a deuterated product ion at m/z 107 (data not shown). The

fragmentation process, shown schematically in Fig. 2, involves a

proton transfer to generate a stable carbonium ion, which is

apparently specific for a-amino acids. Other fragments seen in

the product ion spectrum of methionine include mIx 41, mIx 56,

md m/z 61. The ion at mIx 61 is a fragment incorporating the

ulfur atom (CH3-S-CH2)� (Fig. 1). This fragmentation was

iso observed for d�-methionine with detection of a product ion

t mlz 64 (CD3-S-CH2).

Because methionine exhibits a neutral loss of 102 Da from

hie molecular ion in common with other amino acids whose

agmentation has been described previously [7, 8], the tandem

‘iass spectrometer is set up such that MS1 and MS2 are

ynchronized to scan the mass range (mIx 125-300) with a

onstant 102-Da difference. Product ions that differ by 102 Da

140 160 160 200 22U 240

Mass / Charge

Fig. 1. (Top) Product ion mass spectra from the collision-induceddissociation of [M + Hl� ions of methionine (m/z 206) to smallerpositively charged ions (e.g., m/z 104); (bottom) mass spectra for

neutral loss of 102 Da [M + H -102 1 from methionine in the

tandem mass spectrometer.For a description of the fragmentation processes, see Fig. 2. The common102-Da difference between the original IM + H]� ion and its respective fragmentions is detected by the scan function shown in the lower panel.

from the parent ion of a-amino acids are detected in MS2. Note

that the mass of the parent ion is shown on the mass spectra

although the product ion is what is actually detected. Fig. 1

shows the neutral loss spectrum of the molecular ion (precursor

ion) for pure methionine. In biological samples, the neutral loss

scan detects not only methionine, but also most other common

amino acids (e.g., phenylalanine, tyrosine, leucine, and valine, as

described previously [7, 8]).

Figure 3A shows an amino acid profile from a fresh screening

+ CH2CH2SCH3H3N - C - COOC4H9

CID HCOOC4H9

+

H2N = #{231}-CH2CH2SCH3

H

Fig. 2. Schematic representation of the specific fragmentation in the

tandem mass spectrometer that characterizes amino acid butyl ester

derivatives.

HCOOC4H9 is butyl formate, a neutral fragment of 102 Da common to thefragmentation of the EM + H]’ ions of methionine. CID, collision-induceddissociation.

tOC

C

C0

A

Leu+lIe

Fresh Control

*

100 Pro

1*

*

(0C4)C

c 500

aa)

BAge Matched Control

*Ala

*

eu#{247}lIe

VaI

Sor

(0Ca)C

C0

a)>a4)

180 200 220

Mass / Charge

352 Chace et al.: MS-MS of homocystine in blood spots

Glu

Tyr

140 160 180 200 220 240 260 280 300

Pro Mass/Charge

CHomocystinuria

*Tyr� �*

�240 260 280 300

Fig. 3. MS-MS amino acid profiles obtained by the neutral loss of

1O2�Da scan function used with: (A) a recent screening card from an

unaffected neonate, (B) a 3-year-old screening card from an age-

matched unaffected neonate, and (C) a 3-year�old screening card from

a neonatal patient with homocystinuria.

The asterisks represent deuterated amino acid internal standards. For quantifi-cation the peak height of the amino acid of interest is compared with that of itsrespective internal standards. Each spectrum is normalized to the largest ionsignal present.

card from an unaffected newborn, obtained by using the neutral

loss of 102 Da scan function. Ion signals at representative masses

of several amino acids and internal standards include mlz 206

(methionine) and mlz 209 ([2H3]methionine). The only signif-

icant signals detected are from a-amino acids and their added

internal standards. Fig. 3B shows an amino acid profile from an

unaffected newborn whose screening card had been stored for 3

years. This control is age matched with the newborn from

whose specimen Fig. 3C was derived. Fig. 3C is an amino acid

profile from an original dried blood specimen from a newborn

patient subsequently diagnosed with homocystinuria; the spec-

imen was recovered from storage after 3 years. The ion signal of

methionine in this sample exceeds that for the internal standard

(i.e., mlz 206 relative to mlz 209), as compared with the ion

signals of methionine shown in both Fig. 3A and B.

ASSAY CALIBRATION AND LIMITS OF DETECTION

Calibration curves for methionine were generated by using

standard isotope dilution techniques [12]. The ion signals

corresponding to methionine and its respective deuterium-

labeled internal standard were plotted as a function of the

concentration of methionine added to blood. The calibration

curve for added methionine was linear over the concentration

range of 0-500 �.tmoVL, with slope = 0.0105 (SE = 0.000145),

intercept = 0.306 (SE = 0.0312), and r2 = 0.999 (root mean SE

= 0.065). Typical signal-to-noise ratios for endogenous concen-

trations of methionine in fresh blood samples from unaffected

subjects were �8: 1. The estimated detection limit for methio-

nine, based on a signal-to-noise ratio of 3:1, corresponds to a

concentration of 4 j.�mol/L, far below the range (10-90 �moVL)

of physiological methionine concentrations [13].

ANALYTICAL RECOVERY, IMPRECISION, AND ACCURACY

The analytical recovery of methionine added to blood was

determined in triplicate at concentrations of 0, 50, 100, and 200

Mm0VL. The recovery rate (mean ± SD) was 96% ± 4% for

samples not enriched with methionine. At enrichments of 50,

100, and 200 �tmolfL, the recovery rates (mean ± SD) were

95% ± 4%, 93% ± 5%, and 107% ± 10%, respectively.

The analytical imprecision (CV), determined by replicate

analysis of the derivatized product of a single sample, was 5.2%.

Overall precision of the assay was calculated by replicate anal-

yses of the same unaffected blood sample on the same day and

on different days. The within-day (intraday) imprecision was

5.7% for methionine alone, 3.7% for the ratio of methionine to

phenylalanine, and 6.7% for the ratio of methionine to leucine

plus isoleucine (n 10). The CVs determined over a period of

2 weeks were 7.4% for methionine, 5.4% for the ratio of

methionine to phenylalanine, and 6.3% for the ratio of methi-

onine to leucine plus isoleucine (n = 10). The estimated

concentration of methionine in these samples was 18 .tmoVL.

To compare methionine quantification by MS-MS with

methionine quantification by HPLC, we assayed dried blood

specimens that contained various concentrations of added me-

thionine. These results (Table 1) by both HPLC and MS-MS

represent the total concentration of methionine in blood spots,

i.e., endogenous methionine plus methionine enrichments. The

methionine-enriched samples were made from a single batch 0:

whole blood that contained lysed erythrocytes; the calibration

curve for the MS-MS analysis was generated by using blood

spots prepared from blood with intact erythrocytes. The CDC

estimates that two 3/16-in, disks taken from blood spots pre

pared from lysed-cell blood contain 12.9 �L of blood; w

estimate that two 3/16-in, disks taken from blood spots prepare

from intact-cell blood contain 15.2 �L of blood. When thi

matrix-related difference in blood volumes is taken into account

the values reported by MS-MS closely approximate the addem

methionine at concentrations from 0 to 670 �molJL.

Table 1. Methlonine analysis by tandem MS and HPLC

compared.Enriched Meta Met by MS-MS Met by HPLC

mg/L pmoi/L mg,/L �tmoiIL m&’L �sn1oIIL

0 0.0 2 13.4 4 26.8

20 134.2 18 120.8 16 107.4

40 268.4 35 234.9 31 208.0

50 335.6 53 355.7 47 315.4

50 335.6 43 288.6 35 234.9

50 335.6 54 362.4 49 328.8

60 402.7 50 335.6 39 261.7

70 469.8 59 395.9 49 328.8

80 536.9 68 456.3 55 369.1

100 671.1 92 617.4 70 469.8

� Enrichments prepared with whole blood containing lysed erythrocytes. These

samples contain less blood per disk than intact cells (12.9 j.�L for lysed cells vs

15.2 �L for whole blood).

Clinical Chemistry 42, No. 3, 1996 353

Specimens autoclaved at newborn screening and before storage.

ANALYSIS OF BLOOD SPECIMEN COLLECTIONS

More than 16 000 fresh dried blood spots have been analyzed at

Duke University Medical Center as part of a pilot screening

program in collaboration with the North Carolina State Screen-

ing Laboratory. The majority of these samples were obtained

from the population in the Raleigh-Durham-Chapel Hill re-

gion of North Carolina. In a sampling of this group of neonatal

blood spots, the mean ± SD for methionine was 19 ± 8.3 (n =

909) �tmol/L. This is comparable with the concentration range

for methionine of 10-90 .tmol/L [13] in unaffected neonates.

The value of methionine in our study at 5 SD above the mean,

60.5 �imoVL, also compares well with this range. The methio-

nine to phenylalanmne ratio was 0.39 ± 0.11 and the methionine

to leucine plus isoleucine ratio was 0.16 ± 0.04.

Results of the blinded analysis of dried blood spots received

from the NERNSP are presented in Table 2. This collection

includes six control samples from unaffected patients, four

samples from patients diagnosed with homocystinuria by the

Table 2. QuantItative analysis of methionine in blood spots.

Met, Met:Sample Storage time �emo1IL Met:Phe Leu + lie

Homocystinuria 3.8 years 25.1 0.84 0.27

Control “ 8.6 0.24 0.10

Control “ 9.9 0.26 0.10

Homocystinuria 4.6 years 24.4 0.66 0.29Control “ 9.9 0.60 0.09

Control “ 7.2 0.22 0.10

Homocystinuria 3.4 years 61.7 2.09 0.78

Homocystinuria#{176} 3.5 years 8.6 1.65 0.34

Hypermethioninemia 1.2 years 26.8 0.81 0.37

Hypermethioninemia 40 days 117.0 2.12 1.36

Control 40 days 38.5 0.58 0.28

False positive5 54 days 16.6 0.51 0.17

False positive8 122 days 24.8 1.21 0.17

False positive8 52 days 19.7 0.52 0.22False positive8 54 days 24.0 0.73 0.21

bacterial inhibition assay, four samples from patients with

isolated hypermethioninemia, and four samples in which methi-

onine was measured as increased by the bacterial inhibition assay

but were found to be normal by repeat analysis or follow-up of

false-positive results. The methionine concentrations and the

molar ratios of methionine to phenylalanmne and methionine to

leucine plus isoleucine presented in Table 2 show that the

Met:Leu + lie ratio successfully discriminated between the

positives and both the controls and the false positives. These

data do not allow the prediction of a normal cutoff value for the

Met:Leu + lie ratio because (a) the concentration of methionine

is higher in fresher samples, and (b) the ratio of Met:Leu + lie

is also slightly higher, presumably because of small differences in

their rates of degradation. However, regardless of the storage

age of the samples, in each instance the ratio of Met to Leu +

lie is clearly higher than this ratio in the age-matched controls

(by >2.5 times). Some variabilities in the data are observed,

presumably because the storage times for the specimens vary

from 40 days to >4 years. Notably, some specimens had

previously been autoclaved at the NERNSP laboratory, but no

alterations in the amino acid profiles were apparent (data not

shown).

DiscussionTandem mass spectrometry is a new analytical tool for neonatal

screening of amino acid metabolic disorders. Samples are pre-

pared by a simple semiautomated extraction and derivatization

procedure in batches of 60. Each analysis takes <3 mm. This

method incorporates isotope dilution techniques that provide

accurate quantitative information for specific components of

each sample. The method is robust and capable of analyzing

large numbers of samples because its nonchromatographic na-

ture and new methods of ionization make full automation

possible.

Quantitative data for > 16 000 blood samples from newborns

revealed that the mean concentration pius 5 SD was within the

published upper range for methionine in full-term and prema-

ture newborns. Samples that had been previously categorized as

falsely positive gave normal Met:Leu + lie ratios by MS-MS.

Blinded analysis of samples from six patients previously diag-

nosed with either homocystinuria or hypermethioninemia de-

tected an above-normal Met:Leu + lie ratio in all six (Met:Leu

+ lIe >0.2 7). Of these samples, two had a methionine concen-

tration >67 /LmoVL (>10 mg/L), the cutoff concentration used

for the bacterial inhibition assay in the NERNSP. Because

methionine is easily lost through oxidation, one would expect to

find reduced concentrations of methionine in samples stored as

long as 3 years. The degradation rate of leucine seems closer to

that of methionine than of phenylalanine [14]. Consequently, a

Met:Leu ratio should better reflect the original ratio of these

amino acids than does the Met:Phe ratio and thus allow for the

retrospective detection of hypermethionmnemias (including ho-

mocystinuria). This may explain why the Met:Phe ratio was not

as accurate in detecting homocystinuria. In fresh samples,

however, the Met:Phe ratio might be as valid an index for

differentiating controls, false positives, and true positives for

hypermethioninemia as the Leu + Ile:Phe ratio is in detecting

354 Chace et al.: MS-MS of homocystine in blood spots

maple syrup urine disease [8]. This can be seen by examining the

fresh hypermethioninemia sample after storage for 40 days, for

which the ratio of Met:Phe or Met:Leu + Tie is clearly greater

than that for the age-matched control.

We have no direct data demonstrating the reduction of

false-negative results, having been unable to obtain such sam-

ples. However, the results reported here suggest that the

probability of a false-negative result is greatly reduced. Support

for this assumption is provided in Table 2 for samples in which

the actual concentration of methionine is below the detection

limit while the ratio of Met:Leu + lie exceeds that for the

controls. Further, in a preliminary report, we demonstrated that

the use of molar ratios of amino acids reduced false-positive and

false-negative results attributable to laboratory error or sample

age [15].Our experience shows that MS-MS increases the specificity

of newborn screening for hypermethioninemia. The sensitivity

for methionine quantification by MS-MS appears to be very

good, and its accuracy is indicated by its ability to discriminate

false positives from true positives and by the high correlation of

its results with those obtained by HPLC, a standard assay for

quantifying methionine.

Concerns have been raised by other clinical scientists with

regard to the validity of newborn screening for hypermethi-

oninemias including homocystinuria. These concerns deal with

the prevalence of this disorder in whole-population screening,

the ability to effectively treat homocystinuria, and the cost-

benefit of screening for this disorder. Although these consider-

ations are important, we consider them not particularly relevant

to this paper, which describes and validates the methionine

component of a method that can identify several metabolic

disorders (including phenylketonuria) rather than presenting

data for or against the validity of newborn screening for

homocystinuria. However, the reality is that >20 states and

most European countries and Japan screen for homocystinuria

with a single test that measures methionine concentration;

screening for this disease is often mandated by state legislative

bodies. The new technique we present has greater precision and

accuracy and the ability to diagnose additional diseases screened

for in these same states (phenyiketonuria and maple syrup urine

disease). We believe that MS-MS is entirely cost effective

because it screens for more than one disease in one assay. Using

MS-MS to screen for other diseases such as phenylketonuria and

maple syrup urine disease incurs no additional costs in screening

for homocystinuria. In addition, whole-population screening

with a more precise method than that currently available may

provide important data on disease frequency. Current methods

used to screen for homocystinuria may not be adequately

accurate and precise to detect small increases of methionine in

newborns whose specimens are collected at 24 h or less post-

partum, a result of increasing early discharges of newborns. One

way to address this issue is to develop new, more accurate and

precise methodologies such as the MS-MS method presented

here.We believe that MS-MS applied to newborn screening is a

cost-effective methodology for the following reasons: It allows

for many disease markers to be tested at one time by a single

method, thus substantially expanding newborn screening cover-

age. There are no required kits, and the costs of reagents and

internal standards are minimal. Automation of sample prepara-

tion, analysis, and interpretation, all of which are currently

under development, will increase the number of samples ana-

lyzed per instrument per day, further reducing the cost per test.

The accuracy and precision of the MS-MS method should result

in very low false-positive rates compared with those obtained by

existing methods. Basing diagnosis on the ratios of key metab-

olites reduces the adverse effect of sample collection and prep-

aration on analytical accuracy, as described previously [13]. This

should reduce the number of repeat specimens required and

thus reduce the overall costs of newborn screening.

We are indebted to Susan Moore of the New Hampshire

component and Jane Simmons of the Massachusetts component

of the NIERNSP for recovery of these newborn specimens from

storage. Financial support was from the North Carolina Depart-

ment of Environmental Health and Natural Resources, Division

of Maternal and Child Health, Raleigh, NC (grant no.

C-05070).

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