A Quantitative Method for the Measurement ofDried Blood Spot Amino Acids UsingUltra-Performance Liquid Chromatography

Kaitlyn Bloom,1 Gail Ditewig Meyers,1 and Michael J. Bennett1,2*

Background: Measurement of amino acids in dried blood spots has been extensively used for thedetection of newbornswith various inborn errors of amino acidmetabolism including phenylketonuria(PKU) andmaple syrup urine disease (MSUD). Whereas blood spot amino acid measurement has beeninvaluable for initial diagnosis, the relative insensitivity of blood spot measurement has found limiteduse in lifelong monitoring of patients with these disorders. The work described here outlines theevaluation of blood spot amino acid analysis using ultra-performance liquid chromatography (UPLC©)for use in follow-up testing.Methods: Dried blood spot amino acids were derivatized with a proprietary AccQTag® reagent andseparated using UPLC. Plasma amino acids from dried bloods spots were obtained from 318 patientsamples and compared to corresponding plasma samples measured using the same UPLC method.Results: Dried blood spot amino acid concentrations were highly correlated but negatively biased vsplasma concentrations. Interassay imprecision studies using UPLC demonstrated a %CV for phenylal-anine of 4.81%–16.07%, tyrosine 5.62%–20.16%, valine 4.23%–15.46%, leucine 8.3%–15.3%, and isoleu-cine 4.25%–16.80%. Intraassay imprecision studies using UPLC demonstrated a %CV for phenylalanineof 0.42%–3.4%, tyrosine 1.6%–7.85%, valine 0.14%–1.84%, leucine 0.28%–2.01%, and isoleucine 0.6%–2.65%. Blood spot amino acid concentrations were stable for at least 3 days at temperatures up to65 °C.Conclusions: This UPLC-based method can reliably measure clinically significant amino acids in driedblood spots.

IMPACT STATEMENTPatients with amino acid disorders will benefit from the information presented here. Evidence

presented on blood spot amino acid analysis will allow better characterization of patient manage-ment. Knowledge in the field of pediatric andmetabolic diseasewill be advanced by the informationpresented.

1Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA; 2Department of Pathology andLaboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA.*Address correspondence to this author at: Department of Laboratory Medicine, Children's Hospital of Philadelphia, Palmieri MetabolicDisease Laboratory, 3401 Civic Center Blvd., Philadelphia, PA 19104. Fax 215-590-1998; e-mail bennettmi@email.chop.edu.DOI: 10.1373/jalm.2016.020289© 2016 American Association for Clinical Chemistry

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The measurement of amino acids in dried bloodspots has been extensively used for the detection ofnewborns with various inborn errors of amino acidmetabolism including phenylketonuria (PKU)3 andmaple syrup urine disease (MSUD). Dried bloodspots are a convenient tool for the diagnosis of cer-tain inborn errors ofmetabolism; however, their util-ity in regular monitoring of amino acids has beenlimited. Patients with PKU, MSUD, and some of theurea cycle defectsmust have frequentmonitoring ofplasma amino acid levels to determine the effective-ness of therapy. Treatment for disorders of aminoacidmetabolism involves avoiding the toxic effects ofdietary protein, while at the same time allowing suf-ficientprotein intake fornormal growthanddevelop-ment. This process typically uses specialized proteinor amino acid restrictive diets (1). These patients re-quire careful monitoring of protein intake for propergrowth, which changes with age, development, andother factors (2). Most patients are currently moni-tored using plasma samples with amino acid mea-surement by ion-exchange chromatography, aprocess that can take up to 2 hours per sample.Many of the patients whom we monitor live severalhours away from thehospital and findgreat inconve-nience to travel this distance for routine monitoringpurposes.Whereas blood spot amino acid measurement

has been invaluable for initial diagnosis, the issuesconcerning separation of branched-chain amino ac-ids in blood spot measurement using other technol-ogies has found limited use in lifelong monitoring ofpatients with these disorders (3). We have previouslyfound that ultra-performance liquid chromatogra-phy (UPLC©) can provide a more rapid turnaroundtime foraminoacidanalysis (a run timeof45minutescompared toover2hoursper sample) andhave rou-tinely implemented this procedure in our laboratoryfor plasma amino acid analysis with successful per-formance in the College of American Pathologists(CAP) proficiency program (4).

Previously reported methods of measuringamino acid levels in dried blood spots use technol-ogies such as tandem mass spectrometry (MS/MS), LC-MS/MS, and UPLC-MS/MS (2, 3, 5–7).MS/MS without column separation is used ondried blood spots in newborn screening (3). GC-MShas had little success in quickly and accuratelyquantitating amino acids from dried blood spots(8). UPLC-MS/MS and LC-MS/MSmethods have im-proved the determination of branched-chainamino acids such as alloisoleucine with betterquality separation than previous methods usingHPLC (5, 6). The measurement of amino acids indried blood spots by UPLC with precolumn deriva-tization and in a system not coupled to tandemmass spectrometry has not been fully explored.Use of dried blood spots for monitoring certain

amino acidopathies would bemore convenient forboth laboratories and patients. The work de-scribed here outlines our evaluation of blood spotamino acids using an Acquity UPLC system config-ured with a tunable UV detector and MassTrakAAA® detection system.In the present study, we evaluated the perfor-

mance of dried blood spot amino acid analysis forbranched-chain amino acids, phenylalanine, and ty-rosine using aproprietary reagent andUPLC separa-tion. Results were compared with the plasma aminoacid values obtained using split samples and thesame technology. We have analyzed and comparednormal samples and samples from patients withPKU,MSUD, tyrosinemia, andother conditions toob-tain a wide range of amino acid values.

MATERIALS AND METHODS

Instrumentation and materials

Blood samples (1–3 mL) were collected in so-dium heparin tubes and transported to the labo-ratory at room temperature. Whole blood samples

3Nonstandard abbreviations: PKU, phenylketonuria; MSUD, maple syrup urine disease; UPLC, ultra-performance liquid chromatography.

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were spotted onto Whatman™ filter paper (GEHealthcare Bio-Sciences), and plasma was ob-tained by centrifugation at 1900g from the remain-ing sample. All plasma and dried blood spotsamples were analyzed simultaneously usingUPLC. Sonication was performed using a Sym-phony Ultrasonicator (VWR International). Thefollowing reagents were used in performing thisassay including acetonitrile, Optima® grade(Fisher), methanol, Omnisolv (EMD Chemicals Inc.),andOptima LC/MS gradewater (Fisher). An AcquityUPLC system configured with a tunable UV detec-tor and MassTrak AAA solution kits were obtainedfrom Waters Corporation. The Waters solution kitprovided all of the necessary reagents and con-sumables and has been previously described.

Sample preparation

A total of 318 samples from patients were col-lected for this study. For each sample, we collectedwhole blood and spotted the filter paper cards forthose samples requesting amino acid testing. Thefilter paper was dried overnight at room tempera-ture. Two 6-mmpuncheswere obtained fromeachblood spot for analysis. Each punch contained ap-proximately 12μL ofwhole blood volumewith a totalapproximate volumeof 24μL obtained from the two6-mm punches. The volume of blood contained onone 6-mmpunchwas determined by visual compar-ison of whole blood pipetted onto a spot using stan-dardized volumes. The plasma volume used in theUPLC amino acid procedure was 10 μL.The dried blood spot cards were stored sepa-

rately in envelopes and in plastic bags at 4 °C in astorage container lined with desiccant until time ofextraction and testing. Additional dried blood spotcards were exposed to three temperature condi-tions, including room temperature (21 °C), cold(4 °C), and warm (65 °C) for 3 days. After that, sam-ples were stored at room temperature or 4 °Covernight in a plastic container until time of testingthe following day, to simulate the effects of alteredtemperature exposures during shipping.

Dried blood spot assay

Two 6-mm spots were finely cut into smallerpieces and added to 300 μL of 100% methanol.The sample was sonicated for 1 h and then centri-fuged for 3 min at 13 000 rpm (16 000g). The elu-ent was transferred to a new, labeled Eppendorftube and dried at 40 °C under a stream of nitro-gen. The dried sample was reconstituted in 50 μLof 50:50 (v:v) acetonitrile:HPLC grade water. Thesamples were then placed in a heat block at 37 °Cfor 20 min to aid in reconstitution of the samples.The derivatization of standards, plasma speci-

mens, and blood spot extracts took place in theTotal Recovery Vials supplied with the MassTrakassay kit. Standard derivatization was completedas follows: 10 μL of a 250 μmol/L working aminoacid standard mix was added to 70 μL of a stockborate buffer in a recovery vial. The vial wascapped and vortex mixed thoroughly for 10 s. Atotal of 20 μL of the MassTrak derivatization re-agent was then added to the vial. The vial was re-capped, and the vortex wasmixed thoroughly. Anybubbles were removed from the conical portion ofthe vial before incubation for 10 min at 55 °C.Plasma specimens (100 μL) were deproteinated

with 100 μL of 10% sulfosalicylic acid containing250 μmol/L norvaline (internal standard). The mix-ture was vortex mixed thoroughly and centrifugedfor 3 min at 13 000 rpm (16 000g) in a microcentri-fuge. A total of 20 μL of the supernatant (10 μLplasma and 10 μL SSA) was added to 60 μL boratebuffer, with the pH adjusted with 0.5 mmol/LNaOH (754 μL NaOH in 5 mL stock borate buffer).This mixture was derivatized with 20 μL of the de-rivatization reagent and incubated for 10 min at55 °C.Blood spot sample derivatization was con-

ducted using 60 μL stock borate buffer containing42 μmol/L norvaline (internal standard) mixed with20 μL of the reconstituted sample preparation in atotal recovery vial. The sample was capped, andthe vortex was mixed well. A total of 20 μL of the

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Table1.

Interassay

imprecisionof

driedbloo

dspot

samples

usingUPLC.

Phen

ylalan

ine

Tyrosine

Valin

eLeucine

Isoleu

cine

Interassay

%CV

MeanAA

concen

tration,

μmol/L

Interassay

%CV

MeanAA

concen

tration,

μmol/L

Interassay

%CV

MeanAA

concen

tration,

μmol/L

Interassay

%CV

MeanAA

concen

tration,

μmol/L

Interassay

%CV

MeanAA

concen

tration,

μmol/L

SampleID

AABC

-83

15.0

77.6

20.2

43.3

10.1

149.0

9.6

79.3

9.0

47.1

AABC

-167

4.8

37.8

5.6

57.5

6.7

165.6

6.2

46.6

6.4

53.1

AABC

-168

11.5

30.9

16.1

36.9

6.4

62.8

9.2

34.3

7.2

19.0

AABC

-169

12.9

38.1

12.1

88.3

15.5

43.1

15.3

59.7

16.8

12.3

AABC

-170

6.0

30.6

7.4

22.5

9.1

102.8

9.3

56.5

9.0

27.5

AABC

-171

12.2

35.6

12.0

28.7

14.9

51.0

14.9

33.4

15.0

17.2

AABC

-183

11.3

38.0

13.6

45.1

6.5

135.5

6.5

68.0

5.9

38.1

AABC

-184

6.2

40.7

7.8

39.0

4.2

112.8

4.8

63.5

4.3

33.4

AABC

-185

6.2

31.3

9.1

39.5

8.3

96.2

8.3

54.2

8.5

28.5

AABC

-213

16.1

34.4

16.3

28.0

14.1

53.5

12.9

32.8

13.9

16.4

Table2.

Intraa

ssay

imprecisionof

driedbloo

dspot

samples

usingUPLC.

Phen

ylalan

ine

Tyrosine

Valin

eLeucine

Isoleu

cine

Intraa

ssay

%CV

MeanAA

concen

tration,

μmol/L

Intraa

ssay

%CV

MeanAA

concen

tration,

μmol/L

Intraa

ssay

%CV

MeanAA

concen

tration,

μmol/L

Intraa

ssay

%CV

MeanAA

concen

tration,

μmol/L

Intraa

ssay

%CV

MeanAA

concen

tration,

μmol/L

SampleID

AABC

-17

0.4

23.9

4.3

22.6

1.8

70.5

2.0

35.8

2.7

20.0

AABC

-104

1.1

24.0

2.1

26.1

0.4

113.3

0.6

52.5

1.2

24.3

AABC

-127

0.8

13.9

2.1

17.1

1.1

50.0

1.6

26.8

2.0

14.9

AABC

-149

0.6

19.5

1.6

20.0

0.2

82.9

0.3

38.8

0.8

18.8

AABC

-166

3.4

34.5

7.9

48.4

0.1

63.8

1.1

52.5

0.8

25.1

AABC

-180

0.7

49.2

1.7

46.4

0.7

162.7

0.5

113.4

0.7

55.8

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derivatization reagent was added into the targetvial, and the vortex was mixed thoroughly. The tar-get vials were incubated at 55 °C in a heat block for10 min.

After incubation, the standard, control, and sam-ple vials were placed in the appropriate location onthe autosampler. Each injection flowed through aWaters MassTrak AAA 2.1 μm × 150 mm columnfor separation, with the column temperaturemaintained at 43 °C. UV detection at 260 nmwith acycle time of 45 min was done for each injection.The data analysis of the dried blood spot sam-

ples was conducted using a sample dilution factorof 1.0 for both the plasma control and blood spotsamples. The dilution factor ensures the amountof sample injected is equivalent to the amount ofstandard that is injected. It is estimated that the 20-μLblood spot samples used in derivatization is equivalent

Table 3. Recovery of branched-chain aminoacids and phenylalanine and tyrosine fordried blood spot samples using UPLC.

Valine 84.2 ± 22.2%Isoleucine 89.4 ± 11.6%Alloisoleucine 99.9 ± 8.7%Leucine 89.9 ± 14.3%Tyrosine 87.2 ± 12.6%Phenylalanine 96.0 ± 12.0%

Fig. 1. Bland–Altman analysis of phenylalanine (A) and tyrosine (B) shows negative bias between driedblood spot and plasma amino acid analysis.The derivation of the x axis reflects themean of the dried blood spot and plasma sample concentration. Blue circles indicatesamples without a known diagnosis.

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to the 10 μL of plasma used in control preparation,sincewhole blood is roughly 50%plasma.

Inter- and intraassay precision of UPLC

Intraassay imprecision was determined from 5replicates of a single dried blood spot extract (2- to6-mm spots) on the same day. This step was per-formed on 6 different dried blood spot specimens.Interassay precision was calculated after analyzinga single dried blood spot extract (2 -to 6-mmspots)over 5 consecutive days. Analysis was performedon 10 different dried blood spot specimens. Theevaluation period incorporated multiple changesin batches of reagents and columns to represent aworking laboratory schedule.Recovery studies were carried out using 50 μL of

a 100 μmol/L standard amino acid mixture addedto 450 μL of whole blood, spotted onto filter paperand dried overnight. A dried blood spot filter papercontrol was prepared by spotting whole bloodwithout the amino acid mixture. The samples wereprepared by the extraction and derivatization pro-cedures previously described.

RESULTS

Inter- and intraassay precision

Inter- and intraassay precision was calculatedusing normal samples of whole blood spottedonto filter paper. The CV was calculated for phe-nylalanine, tyrosine, valine, leucine, and isoleu-cine. The interassay CV for each amino acid isrepresented in Table 1. The intraassay %CV foreach amino acid is represented in Table 2. Inter-assay imprecision observed with blood spots isslightly higher than that of plasma. This findingmay be due to changes in the environment of thelaboratory or in calibration or simply reflectthe known lack of sample homogeneity acrossthe blood spot.

Recovery of added amino acids

The recoveries of the added amino acid stan-dard mixture for the branched-chain amino acidsand phenylalanine and tyrosine were determinedin triplicate and are presented in Table 3. In

Fig. 2. Bland–Altman analysis of valine (A), leucine (B), isoleucine (C), and alloisoleucine (D) shows amoderate negative bias between dried blood spot and plasma amino acid analysis.The derivation of the x axis reflects themean of the dried blood spot and plasma sample concentration. Blue circles indicatesamples without a known diagnosis.

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general, added amino acids were quantitativelyrecovered.

Comparison of quantitative amino acid databetween plasma and dried blood spots

For phenylalanine and tyrosine, dried blood spotanalysis has a negative, proportional bias vsplasma (Fig. 1). For valine, leucine, isoleucine, andalloisoleucine, dried blood spot analysis also has amoderate negative proportional bias vs plasma(Fig. 2). Linear regression analysis of phenylala-nine, tyrosine, valine, isoleucine, leucine, and allo-isoleucine in blood spots vs plasma are presentedin Fig. 3.Reference interval estimates were also gener-

ated from 185 dried blood spots with normalamino acid profile results selected at random. In-terval estimates were derived from the mean ± 2SDs, since these specimens were not scrupulouslyselected from healthy fasting children. Estimateddistributions for phenylalanine, tyrosine, andbranched-chain amino acids using dried bloodspots are shown in Table 4.To examine the stability of blood spots when

exposed to changes in temperature and storage

conditions, blood spots were exposed to 4 °C or65 °C in an incubator for 3 days, followed by over-night storage at 4 °C or room temperature. Theresults of this study demonstrate that the bloodspot filter papers are stable for at least 3 days de-spite prolonged exposure to increased tempera-ture (Fig. 4).

DISCUSSION

The measurement of amino acids from driedblood spots is a convenient tool for continuedmonitoring of patients with many inborn errors ofmetabolism. This study demonstrates that UPLCwith precolumn derivatization is capable of

Fig. 3. Comparative analysis of phenylalanine, tyrosine, valine, isoleucine, leucine, and alloisoleucinein dried blood spots and plasma using linear regression.

Table 4. Modified reference ranges establishedfor using dried blood spot analysis.

Average SD 2 SDRangelow

Rangehigh

Phenylalanine 45.26 20.37 40.75 4.51 86.00Tyrosine 55.92 22.36 44.71 11.21 100.63Valine 150.24 54.11 108.23 42.02 258.47Isoleucine 49.31 22.80 45.60 3.70 94.91Leucine 86.29 38.86 77.72 8.57 164.01

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detecting amino acid levels extracted from driedblood spot filter papers. Blood spot amino acidconcentrations are highly correlated with plasmaand are stable under conditions that mimic thosethatmight be encountered during transport. Driedblood spots, therefore, are convenient for specialcases where patients may be far from a metaboliccenter, have difficulty obtaining transportation forclinic visits, or are in difficult social situations suchas incarceration.In the Bland–Altman and regression plots, we

observed a modest negative bias for blood spotamino acids when compared to plasma. Thecauses of this bias are not completely clear. Onepossibility may be lower extraction efficiency. It isalso likely that the distribution of amino acids

across red cell membranes may be responsiblefor the observed bias. Finally, due to variability inhematocrit, our estimates of plasma water con-tent in the blood spots may be inaccurate andlead to the observed bias. As a result of this bias,we have modified our reference range estimatesto address these changes. For patients withamino acid disorders, the process remains satis-factory, and therapeutic monitoring can bemade by blood spot analysis with the recognitionof the concentration bias. We plan to use thisapproach as a clinical test for monitoring ourlong-distance patients with PKU and MSUD. Clin-ical management will be determined by the phy-sicians. Other amino acids will be evaluatedusing this system in our future studies.

Fig. 4. Effect of storage temperature on dried blood spot concentrations of phenylalanine (A), tyrosine(B), valine (C), leucine (D), and isoleucine (E).RT, room temperature.

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Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and havemet the following4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b)drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable forall aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriatelyinvestigated and resolved.

Authors’ Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Role of Sponsor: No sponsor was declared.

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