Simple HPLC Method with Internal Standard forEvaluation of Vitamin B1 Status By Use ofWhole Blood

Xiaochun Zhang,1* Xiaoying Tang,1 Bill Gibson, Jr.,1 and Thomas M. Daly1

Background: Two primary assays are routinely used for evaluating a patient's vitamin B1 status: plasma free thiamine and

whole blood thiamine diphosphate (TDP). TDP is the bioactive form of vitamin B1 and best reflects body stores. Plasma free

thiamine levels are driven by recent dietary intake. The objective of this study was to develop a simple HPLC method with an

internal standard (IS) that simultaneously measures TDP and thiamine in whole blood, and to assess the use of this single-tube

assay to provide comprehensive evaluation of vitamin B1 status.

Methods: The final assay used amprolium thiochrome as an IS, and the sample preparation procedure takes approximately

1 h. Whole blood thiamine and plasma thiamine were concurrently measured for 126 subjects.

Results: The analytical measurement range was 1.7 to 442.3 nmol/L (TDP) and 1.7 to 375.4 nmol/L (thiamine), with interassay

precisions of 4.0% to 4.8% (TDP) and 2.9% to 8.0% (thiamine), respectively. Method comparisonwith a reference laboratory HPLC

method showed r = 0.9625, slope = 1.021, and intercept = 0.982 (n = 53) for TDP quantification. Whole blood thiamine correlated

closely with plasma thiamine levels but were slightly higher with amean difference of 1.0 nmol/L (range: −3.0 to 5.0 nmol/L). The

reference interval for whole blood TDP and thiamine was 84.3 to 213.3 nmol/L and 1.7 to 21.9 nmol/L, respectively.

Conclusions: This assay provides a simple and reliable HPLC method with a suitable IS for quantification of both TDP and

thiamine from whole blood. It also eliminates the need for separate samples for TDP and thiamine measurement, which will

allow both short-term and long-term vitamin B1 status to be assessed from a single sample.

IMPACT STATEMENTEvaluating a patient's vitamin B1 status currently requires 2 primary assays: whole blood thiamine diphosphate (TDP) to

assess total body stores and plasma free thiamine for recent dietary intake of vitamin B1. This study provides a simple and

reliable HPLC method with a suitable internal standard for quantification of both TDP and thiamine from a single whole

blood sample. Our data show that free thiamine whole blood levels correlated well with plasma levels, eliminating the need

for separate samples for TDP and thiamine, whichwill allow both short-term and long-term vitamin B1 status to be assessed

from a single sample.

1Department of Laboratory Medicine, Robert Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH.*Address correspondence to this author at: Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195. Fax 216-636-0292;e-mail zhangs@ccf.org.DOI: 10.1373/jalm.2017.024349© 2017 American Association for Clinical Chemistry2Nonstandard abbreviations: TMP, thiamine monophosphate; TDP, thiamine diphosphate; IS, internal standard.

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Copyright 2017 by American Association for Clinical Chemistry.

Vitamin B1 is an important cofactor for enzymesinvolved in amino acid and carbohydrate metabo-lism in humans. Severe vitamin B1 deficiency ischaracterized by cardiovascular (wet) beriberi, neu-rological (dry) beriberi, and Wernicke–Korsakoff syn-drome (1), or, less commonly, lactic acidosis (Shoshinberiberi) (2). In Western countries, vitamin B1 defi-ciency is most commonly seen with chronic alcohol-ism, as well as liver and gastrointestinal diseases,although recent studies have suggested that vitaminB1 deficiency may also play a role in patients withdiabetes, obesity, or heart failure (3–8).Vitamin B1 is a group of 4 vitamers that include

free thiamine, thiamine monophosphate (TMP)2,thiamine diphosphate (TDP), and thiamine triphos-phate. Free thiamine is the major form of vitaminB1 found in both dietary sources and vitamin sup-plements, and is the predominant human plasmaform. TDP is the biologically active form of vitaminB1 that makes up 80% of total circulating vitaminB1 in humans (9). TDP is mainly located in erythro-cytes and exists at negligible levels in plasma.Whole blood TDP levels correlate with whole-bodystores of vitamin B1 and are commonly used as aprimary marker for determining long-term vitaminB1 status (9, 10). Free plasma thiamine levels re-flect recent dietary intake of vitamin B1 and as aresult are less useful in identifying the presence ofpersistent B1 deficiency in patients (11). The phys-iological function of TMP and thiamine triphos-phate remains largely unclear. Findings indicatethat thiamine triphosphate may play a neurophys-iological role via regulating chloride channels (12).TMP and thiamine triphosphate are currently notused in clinical assessment of vitamin B1 status.Assays for evaluating patient vitamin B1 status

have evolved from indirect indicators such as tran-sketolase activity to direct measurement of vitaminB1usingeither LC-MS/MSorHPLC (9,10). Becauseofthe difficulty of TDP ionization, most LC-MS/MSmethods initially convert TDP to thiamine by anover-nightenzymatic reactionusingacidphosphatase (13)followed by measurement of the resulting thiamine

as a surrogate for total TDP. In contrast, HPLCmeth-ods can directly measure TDP levels in whole bloodby oxidizing to fluorescent thiochrome.Because no in vitro diagnostic assays for vitamin

B1 are available, most HPLC methods offered byclinical laboratories are laboratory-developedtests based on either a commercially available re-search use only kit (Chromsystems) or internallydeveloped protocols. The Chromsystems kit usesan internal standard (IS) to control imprecisionbut requires 2 incubation steps at 60 °C and 4 °C,respectively, and has a limited 2-point calibrationcurve (vendor website). Most published laboratory-developed tests (10, 12, 14, 15) use a precolumn ox-idation of B1 vitamers by use of alkaline potassiumferricyanide. However, these laboratory-developedtests lackan ISandneedeitheran ionpairing reagentin separation (16) or a labor-intensive liquid/liquidex-traction with methyl-tert-butyl ether to remove thetrichloroacetic acid used to precipitate protein dur-ing sample preparation (14). Our goal was to developa simple HPLCmethod with an IS that could simulta-neously measure TDP and thiamine in whole bloodwithout using amethyl-tert-butyl ether wash in sam-ple preparation or ion pair reagents in separation.

MATERIALS AND METHODS

Chemicals and reagents

Amprolium hydrochloride, TMP, TDP, and potas-sium ferricyanide were purchased from Sigma-Aldrich. Thiamine HCl (certified reference material)was purchased fromCerilliant. Hydrochloric acid, so-dium phosphate dibasic anhydrous, and trichloro-acetic acid were purchased from Thermo Fisher.Methanol (HPLCgrade)wasobtained fromBurdick&Jackson. Sodium hydroxide solution (50%, HPLCgrade) and phosphoric acid (85%, HPLC grade) wereobtained from FLUKA/Fisher Scientific. Solutionswere filtered through a 0.22-μm filter before use.Waterwas generatedusing aMilliporewater system.

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Preparation of stock solutions and IS

Alkaline potassium ferricyanide solution wasused to oxidize amprolium, TDP, TMP, and thia-mine into their fluorescent thiochrome. Methanolwas used to intensify the fluorescent signal. Thethiochrome reaction mixture was prepared bymixing 1 part of methanol with 2.5 part of 0.04%potassium ferricyanide in 15% NaOH. The alkalinepotassium ferricyanide solution was filtered usinga 0.22-μmol/L cellulose acetate filter before use.The fluorescent thiochrome of amprolium was

used as an IS. To prepare amprolium thiochrome,1-mmol/L amprolium hydrochloride stock solutionwas made by dissolving amprolium hydrochloridein methanol. The stock solution was then dilutedwith water to a 20-μmol/L working solution. Thissolution was mixed with the thiochrome reactionmixture at an 8:7 ratio and incubated at 4 °C for 1 hto create the fluorophore (amprochrome) by aring-closing reaction.A sample treatment mixture was used to precip-

itate protein. The treatmentmixture was preparedby mixing 6.7% Trichloroacetic acid (TCA) solutionwith the IS solution described above at a ratio of79:1.

Quality controls and calibrator preparation

A stock solution of TDP (approximate concentra-tion 1 mmol/L) was prepared in 0.1 mol/L hydro-chloric acid, and the exact concentration of theTDP stock was then determined using a spectro-photometer with molar absorption coefficient of13000 L/mol/cm at 247 nm. A secondary refer-ence standard (1-mg/L solution) obtained fromCerilliant was used to prepare thiamine calibra-tors. Stock solutions were further diluted usingsaline to prepare 5 levels of calibrators. The con-centrations of calibrators are 400, 160, 64, 25.6,and 10.24 nmol/L for TDP and 200, 80, 32, 12.8,and 5.12 nmol/L for thiamine. These concentrationlevels span the range commonly seen in patientsamples.

Two levels of quality controls were prepared us-ing residual patient EDTA whole blood specimens.The high control was generated by pooling sam-ples with vitamin B1 levels close to the upper limitof reference interval. Because vitamin B1-deficientsamples were rare, the low control was preparedby diluting a random patient pool with saline tobelow the lower limit of the reference interval.

Sample preparation

Whole blood samples were collected into EDTA-or heparin-containing tubes. On receipt in the lab-oratory, the specimenswere frozen at −70 °C for atleast 4 h before being analyzed to ensure com-plete lysis of red cells. For analysis, 250 μL of wholeblood thawed at room temperature was addedinto a microcentrifuge tube containing 750 μL ofsample treatment mixture. Saline blank, calibra-tors, and controls were handled in an identicalmanner. All sample tubes were loaded on a vortex-type mixer and vortex-mixed at 3000 rpm for 10min at room temperature. After mixing, the sam-ples were left at 2 to 8 °C for 10 min and thencentrifuged at 15000g for 10 min at 4 °C. Each su-pernatant was transferred into a well of a lipid-removal filter plate (Captiva ND Lipids 96-wellplate, Agilent, Santa Clara, CA) with an attached96-well collection plate to remove any remainingprecipitated protein and lipids. The filtrated super-natants (200 μL) were then transferred to amberglass vials with 175 μL of freshly made thiochromereaction mixture. Thiochrome reaction mixturewas prepared freshly each day and used within 2 hafter filtration. After mixing, the vials were placedon the autosampler for HPLC analysis. The samplepreparation procedure takes approximately 1 h.

Chromatographic conditions

A Vanquish ultra-HPLC system equipped withfluorescence detector (Thermo Fisher Scientific)was used in the analysis. Separation was achievedat 25 °C using an Agilent Poroshell 120 EC-C18

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column (3.0 × 50mm, 2.7 μm). Mobile phase A was25-mmol/L dibasic sodium phosphate (pH 7.0),andmobile phase B was methanol. Gradient stepswere as follows: −2.5 to 0 min, equilibration with97% A (3% B); 1 min, 20% B; 4 min, 40% B; 4.5 min,40% B; 4.6 min, 45% B; 5.5 min, 70% B, and thenreturn to equilibration. Injection volumewas 20μL,and flow rate was 0.6 mL/min. The fluorescentproducts were detected at an excitation wave-length of 375 nm and emission wavelength of 435nm. Themaximum excitation and emission of thio-chrome are at 375 nm and 432 to 435 nm, respec-tively (17). Spectral bandwidth was 20 nm for bothexcitation and emission. Peak area was measured.The length of the analytical run was 5.5 min.

Characterization of assay performance

Assay precisionwas evaluated using the 2 levels ofquality control samples. These samples were as-sayed in duplicate each day with 1 replicate immedi-ately following the calibrators at the beginning of therun and the other replicating by the end of the run.Imprecision was estimated over a total of 20 days.Linearitywasevaluatedbydilutingwholebloodspec-imens spikedwithahigh concentrationof target ana-lytes tospana rangeof1.7 to442nmol/L forTDPand1.6 to 375 nmol/L for thiamine. All linearity sampleswere assayed in triplicate. The lower limit of quantifi-cationwasdefinedas the lowest levelwherebiasandimprecision were both <20%.Spike recovery was evaluated by assaying pa-

tient pools spiked with TDP/thiamine at variousconcentration levels. Recovery was determinedusing the following formula: Recovery (%) = 100 ×(measured concentration of spiked sample − aver-age concentration of saline spiked duplicates)/spiked concentration. Intermethod comparisonwas performed against 2 different reference labo-ratories, 1 using a published HPLC method and 1using an unpublished LC-MS/MS method. In addi-tion, calibrator materials from a commercial kit(Chromsystem) were assayed as unknowns andcompared with the manufacturer's stated value.

Calibrator stability was evaluated by testing ali-quots of the working solution stored at −70 °C atvarious time points. Stability of IS was determinedwhen mixed with 6.7% TCA as part of the sampleprep mixture. Specimen storage stability at roomtemperature, refrigerated, and frozen (−20 °C) wasevaluated using pooled patient samples with lowand high concentrations of vitamin B1. Stability ofposttreated samples was tested using both cali-brators and patient specimens when stored in theautosampler at 6 °C. For all stability studies, peakarea was used for evaluation, with a percentchange of <15% from baseline considered stable.Reference intervals were generated using ran-

dom samples from patient samples submitted forcomplete blood count. Chart review was per-formed, and samples from patients with alcoholabuse, malnutrition, gastrointestinal diseases, ortaking vitamin B supplements were excluded. A to-tal of 128 samples from screened patients weretested for TDP and thiamine to establish ranges.

Correlation between whole blood thiamineand plasma thiamine

To determine whether thiamine levels gener-ated from whole blood would compare well withthe standard approach of measuring thiamine inplasma, residual material from 126 EDTA tubessubmitted for complete blood count testing wascollected. Each sample was split into 2 aliquots.One aliquot was immediately centrifuged at 3000rpm for 20 min, and the resulting plasma was sep-arated for measurement of plasma thiamine. Thesecond aliquot was frozen at −70 °C for 24 h andthen assayed for TDP and whole blood thiamine.

Statistical analyses

Precision, method comparison, linear range, ref-erence intervals, and detection limits were calcu-lated using EP Evaluator software (Release 10).Comparison between 2 groups was determinedusing t-test, and P values <0.05 were regarded as

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statistically significant. Correlation and t-test wereperformed using Sigma Plot (version 11.2). Refer-ence intervals were defined as the central 95% ofthe data for each marker using nonparametric,parametric, or transformed parametric analysisdepending on data distribution.

RESULTS

Impact of potassium ferricyanide concentrationonvitaminB1 thiochrome reaction

Thiochrome reaction conditions were optimizedby comparing potassium ferricyanide solutions

ranging from 0.02% to 0.40% to determine theconcentration that provided the best fluorescentyield (see Fig. 1 in the Data Supplement that ac-companies the online version of this article athttp://www.jalm.org/content/vol2/issue3). At thehighest and lowest concentrations, patient sam-ples and calibrators showed different rates of con-version, leading to a loss of correlation betweenthose sample types. Total fluorescence was stron-gest at a concentration of 0.04% potassium ferri-cyanide, and was chosen as the final conditionmoving forward. In addition, the time course ofthiochrome reaction was evaluated using 0.04%

Fig. 1. Selection of a suitable IS for the vitamin B1 whole blood HPLC method.Chromatogram of pyrithiamine (1000 nmol/L in water) after oxidization with potassium ferricyanide (A). Small amount ofTMP, TDP, and thiaminewas found in the commercially available pyrithiamine. Chromatograms of amprolium (500 nmol/L inwater) after oxidization with potassium ferricyanide (B). Commercially available amprolium contains about 3% impurity, butno vitaminB1was found.Overlay chromatogramsof amprolium in calibrator-saline solution (black) and inwhole blood (blue)after oxidizationwith potassium ferricyanide (C). The amproliumpeak areawas significantly greater in salinematrix than thatin whole blood. Overlay chromatograms of preformed amprolium thiochrome in calibrator-saline solution (black) and inwhole blood (blue) after oxidization with potassium ferricyanide. The amprolium peak area was equivalent between salinematrix and whole blood matrix (D).

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potassium ferricyanide. The peak area of TDP thio-chrome was the highest immediately after mixingTDP with the thiochrome reaction mixture andthen remained stable for at least 24 h (see Fig. 2in the online Data Supplement). The results indi-cated the thiochrome formation completed withinminutes after adding the reaction mixture.

Selection of a suitable ISThree chemicals that were structurally similar to

thiamine and produced fluorescence after alkalinepotassium ferricyanide oxidation were evaluatedas potential IS. Acetylaneurine comigrated with thi-amine when using the C18 column and could notbe separated by optimizing HPLC conditions (data

Fig. 2. Matrix choice for calibrator solution.All chromatograms are fluorescence response after oxidation with potassium ferricyanide. The top panel shows saline as anacceptablematrix for preparing vitamin B1 calibrators. Chromatogramof saline blank showing it is free of vitamin B1 (A). 400nmol/L of TDP in saline, <3% was converted to TMP (B). TDP remains stable for at least 4 h in saline (C). The middle panelshows TDP is unstable in 5% BSA solution, and a significant amount of TDP converted to TMP. 5% BSA solution does notcontain detectable amount of B1 (D). About 14%of TDP converted to TMP shortly after TDPwas added to the 5%BSA solution(E). About 75% of TDP converted into TMP after 4 h in 5% BSA solution (F). The bottom panel shows TDP was unstable instripped plasma. (G), Charcoal stripped plasmawas free of vitamin B1. About 39%of TDP converted to TMP after it was addedto the plasma (H). IS: amprolium thiochrome. RT, room temperature.

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not shown). Pyrithiamine showed an ideal reten-tion time with a peak between the TDP and thia-mine peaks. However, commercially availablepyrithiamine from either Sigma Aldrich or TorontoChemicals contained small amounts of thiamine(Fig. 1A). Amprolium showed an acceptable reten-tion time and did not show any contaminants af-fecting thiamine or TDP peaks (Fig. 1B). However,the fluorescent signal generated by amproliumwhen spiked directly into whole blood sampleswas significantly different than that seen incalibrator-saline solutions (Fig. 1C). This problemwas resolved by first oxidizing amprolium to its flu-orescent thiochrome and then using the pre-formed amprolium thiochrome as the IS (Fig. 1D).The CV of amprolium thiochrome peak area wastypically between 2.8% and 4.4% within eachrun.

Selection of matrix for calibrator solution

Initial attempts were directed at producing ma-trices based on whole blood, which would requireremoval of the physiologic levels of TDP and thia-mine present. Multiple methods including UV ex-posure and charcoal stripping were unsuccessfulat reducing vitamin B1 to undetectable levels inwhole blood specimens (data not shown). We nextevaluated alternative matrices including saline, 5%BSA, and commercially available charcoal strippedplasma (Fig. 2). No background TDP or thiaminewas detected in any of these matrices. However,TDP spiked into either 5% BSA or charcoal-stripped plasma showed gradual conversion intoTMP over very short periods (Fig. 2). In contrast,TDP remained stable in saline solution for at least4 h when kept in dark and on ice. Saline was se-lected as matrix to prepare calibrator solutions.

Performance characteristics

The representative chromatograms and typicalstandard curves of TDP and thiamine were shownin Fig. 3. The analytical performance characteristics

of the assay are summarized in Table 1. Using lipid-removal filter plate reduced the CV of IS peak areafrom an average of 25% to <5% within each runand greatly improved the assay precision. Stabil-ity of specimens, calibrators, and IS are listed inTable 1. TDP was stable in whole blood samples,and no degradation or conversion to TMP wasobserved at various temperatures during thetime frame specified in Table 1. Reference inter-vals for TDP and thiamine were established usingwhole blood specimens from 128 nonalcoholicindividuals who were not taking vitamin B1 sup-plements. The reference population included 75women and 53 men with ages ranging from 18 to94 years. No significant difference was found be-tween sexes (P > 0.05). Whole blood TDP/thia-mine levels were 138.9 (33.9)/6.8 (5.8) nmol/L inwomen and 136.7 (36.2)/5.2 (4.0) nmol/L in men,respectively. The established reference intervalis listed in Table 1. The median of TDP and thia-mine in whole blood was 132.7 nmol/L and 4.4nmol/L, respectively.Assay results showed good agreement to a

reference laboratory HPLC method (Fig. 4), andthe measured value of a commercially availablecalibrator (Chromsystems) showed <5% differ-ence from the manufacturer assigned value(92.6 nmol/L). However, a subset of specimensshowed a positive bias when compared with anLC/MS-MS method in a reference laboratory.Further investigation of this subset showedthese samples to have high levels of free thia-mine. To investigate whether these levels mightbe causing an interference in 1 of the assays,we prepared a pooled whole blood specimenspiked with either 200 nmol/mL or 2000 nmol/mLthiamine. Measured TDP values were unaffectedby high thiamine levels using the in-house HPLCmethod. However, reductions of 19% to 21% inTDP measurements were seen in the spiked sam-ples when tested using the reference laboratoryLC/MS-MS (Fig. 4).

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Correlation between plasma thiamine andwhole blood thiamine

We next compared the thiamine levels betweenwhole blood andplasma from the same individuals

(n = 126). A strong correlation and linear relation-ship was observed between the thiamine levelsgenerated from either plasma or whole blood (R2 =0.9082, slope = 1.0134, intercept = 0.8803; Fig. 5).

Fig. 3. Representative chromatograms of vitamin B1 and standard curves.All chromatograms are fluorescence responses after oxidation with potassium ferricyanide. Overlay chromatograms TDP,TMP, thiamine, and IS from 5 levels of calibrators in saline (A). A typical standard curve of TDP (B). A typical standard curve ofthiamine (C). Chromatogramof a patient whole blood samplewith normal level of TDP (159.2 nmol/L) and high concentrationof thiamine (>375 nmol/L), consistent with taking vitamin B1 supplement (D). Chromatograms of a patient whole bloodsamplewith vitaminB1deficiency (TDP, 63nmol/L; thiamine, 4.4 nmol/L) (E). IS: amprolium thiochrome. The yellowdiamondsindicate the highest calibrator.

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Whole blood thiamine levels were slightly higherthan plasma thiamine (medians of 4.6 and 3.7nmol/L, respectively; P = 0.015). The average differ-ence between whole blood and plasma thiaminewas 1.0 nmol/L (range, −3.0 to 5.0 nmol/L). No lin-ear relationship was observed between TDP levelsand thiamine levels.

DISCUSSION

Vitamin B1 plays an important role in metabolismand neurologic function, and assays to evaluate po-tential deficiency are used in many diagnostic work-ups. Although vitamin B1 deficiency has mostcommonly been associated with chronic alcoholabuse, lesser degrees of deficiency have beendescribed as contributors to a broad spectrum ofdiseases, such as diabetes, obesity, heart failure, de-

mentia, and cancer (4, 5, 18–20). The assessment ofvitamin B1 status can be somewhat challenging forlaboratories because of the lack of high-throughputautomatedassaysandcommercial in vitrodiagnostickits. For laboratorieswith thecapacity toperform lab-oratory-developed tests, HPLC and LC-MS/MSmeth-ods are commonly used to measure TDP andthiamine. However, these assays often require longincubations or complex extraction protocols. The as-say described in this report provides an optimizedHPLCmethod that provides several advantages overcurrent methods, including a simplified extractionprotocol, an IS to improve assay precision, and theability to measure both TDP and thiamine from asingle sample.Both cyanogen bromide and potassium ferricya-

nide oxidation methods have been used in detec-tion of thiamine and its phosphate esters. The

Table 1. Assay performance characteristics.

Assay precision

Analytes

Low control High control

Mean(nmol/L)

CV%Intraassay

CV%Interassay

Mean(nmol/L)

CV%Intraassay

CV%Interassay

TDP (n = 40) 66.6 2.3 4.8 161.0 2.5 4.0Thiamine (n = 40) 2.9 5.0 8.0 12.8 3.5 2.9

Analytical measurement range and reference interval

Analytes AMR Reference intervalTDP 1.7–442.3 nmol/L 84.3–213.3 nmol/LThiamine 1.7–375.4 nmol/L 1.7–21.9 nmol/L

Spike recovery

TDP Mean recovery: 101.5% Range: 98.8–105.1%Thiamine Mean recovery: 96.8% Range: 90.1–110.9%

Specimen and reagent stability

Ambient Refrigerated FrozenWhole blood specimen 8 h 7 days 6 months (−70 °C)Postoxidization specimen 3 daysCalibrators (work solution) 3 months (−70 °C)Calibrators (stock solution) 1 year (−70 °C)IS 2 months in 6.7% TCA 2 months in 6.7% TCA 6 months without TCA (−70 °C)

AMR: Analytical measurement range; TCA: Trichloroacetic acid.

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latter offers several advantages, such as low toxic-ity, fast reaction speed, and reaction occurring atrefrigerated conditions. However, the concen-tration range of potassium ferricyanide thatgives an optimal thiochrome yield is narrow. Toolittle potassium ferricyanide gives a low thio-chrome yield, whereas an excess amount alsodiminishes the production. A 0.04% potassium

ferricyanide alkaline solution prepared by weigh-ing 10 mg of potassium ferricyanide with an an-alytical balance (0.1-mg readability and 0.1-mgrepeatability) and then adding 25 mL of 15% so-dium hydroxide solution with serological pi-pettes (accuracy within ±2%) is practical andprovides sufficient precision. Methanol is in-cluded in the thiochrome reaction mixture to

Fig. 4. Method comparison of TDP inwhole bloodwith a reference labHPLCmethod and a reference labLC/MS-MS method.Comparison of the in-house HPLC method with a reference lab HPLC method showed equivalent measurement of TDP inwhole blood specimens (A). Biaswas observed for a subset of specimensbetween the in-houseHPLCmethodanda referencelab LC/MS-MS (B). The correlation coefficient was 0.9625 and 0.9530 for the HPLC and LC-MS/MSmethods, respectively. Highconcentration of thiamine in samples adversely affected TDP measurement by the LC-MS/MS method but not the in-houseHPLC method. The black dashed lines in (A) and (B) indicate the 1:1 line (C).

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intensify the fluorescent signal and enhance stabil-ity of the fluorescent products (14).Finding a suitable IS was challenging. A workable

IS needs to generate fluorescence, resist strongacid used in the protein precipitation step, andmi-grate with a retention time compatible with theTDP and thiamine molecules. Although acetylan-eurine was used in an assay for TDP in washed andlysed erythrocytes (21), our study showed that theC18 column could not separate acetylaneurineand thiamine and therefore would not work for anassay that measures both analytes. Pyrithiaminewas used as an IS in measurement of B1 in cellculture media by ion pair HPLC (16), and has anideal retention time between TDP and thiamine.However, the commercially available pyrithiaminewe tested contained small amounts of TDP andthiamine, which could interfere with accuratemea-surements. The IS we settled on (amprolium) ful-

filled the initial requirements of fluorescence,resistance to acid, and appropriate migration.However, a key finding was that the thiochromeformation rate of amprolium in saline-based cali-brators differed from whole blood samples, mak-ing it unsuitable for use directly. This was correctedby oxidizing amprolium into its thiochrome beforeuse as an IS. The amprolium thiochrome performswell as an IS and is stable at various storage condi-tions and throughout the sample preparation.The accuracy of this method was evaluated us-

ing a commercially available calibrator and by com-parison with HPLC and LC-MS/MS methodsperformed at reference laboratories. Correlationwith the HPLC method was good across the fullrange of samples tested, even though the HPLCmethod being used at the reference laboratoryused a slightly different protocol (which included amethyl-tert-butyl ether wash procedure and lackof IS). This indicates that harmony among HPLCmethodologies can be achieved regardless of thespecific thiochrome reaction method or samplepreparation processes. Our method also corre-lated well with an LC/MS-MS method measuringTDP for the majority of samples. However, a smallpositive bias was observed in a subset of samplesthat contained high levels of free thiamine, whichwas further demonstrated using spiking studies.One possible mechanism for this may be that be-cause the LC/MS-MS method converts TDP to freethiamine by an enzyme reaction before measure-ment, the presence of high thiamine could poten-tially inhibit the conversion and lead to low TDPmeasurement.An additional advantage of the assay described

here is the ability to measure both thiamine andTDP from whole blood, which allows clinicians toassess both short-term and long-term vitamin B1stores using a single specimen. Our data demon-strate that whole blood thiamine highly correlateswith plasma thiamine concentrations measuredfrom the same patient. This is consistent withknown vitamin B1 biology, where erythrocyte

Fig. 5. Correlation between plasma thiamineand thiamine in the whole blood.A total of 126 EDTAwhole blood sampleswere used. Eachsample was divided into 2 parts. One part was frozen at−70 °C for 24 h and then assayed for whole blood thia-mine. The other part was centrifuged to generate plasmaand tested for plasma thiamine. Whole blood thiamine ishighly correlated with plasma thiamine and exhibited alinear relationship with a slope close to 1. The median ofwhole blood thiamine was 4.6 nmol/L, which was slightlyhigher than the median of plasma thiamine, 3.7 nmol/L(P = 0.015).

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thiamine levels are relatively low and thereforeshould not contribute much to the whole bloodlysate. Tallaksen et al. (22) measured the concen-tration of thiamine and its 3 phosphate esters inwhole blood and serum from 30 healthy donors.Although their method used a different oxidationreagent (cyanogen bromide) and did not containan IS, the major findings were consistent with ourresults. They reported comparable thiamine levelsin whole blood and serum. Our assay also containsTMP calibrators and can measure TMP levels. Ourdata show TMP remains at low levels [2.7 (0.3)nmol/L, n = 126] in whole blood and does not cor-relate with either vitamin B1 deficiency or supple-ment intake. Because of its insignificant amountand unclear physiological role, TMP amount is notformally reported in this assay.An important consideration is how best to set a

reference interval for TDP. Many laboratories use70 nmol/L as the lower limit of their reference in-terval (9) based upon the Institute of Medicine'sdefinition of deficiency (23). The recommendeddaily allowance of vitamin B1 is based on thesecriteria. However, the patient-basedpopulationweused for our reference study skewed toward

higher values, with a calculated lower limit of 84nmol/L. Many patients who were excluded fromthe study because of conditions such as gastricbypass surgery, gastrointestinal bleeding, end-stage kidney disease, end-stage malignancy, ormoderate degree of malnutrition often had TDPlevels between 70 and 85 nmol/L, suggesting thatthe “marginal deficiency” range (70–90 nmol/L) de-fined in the Institute of Medicine report may bemore common than expected. Defining the refer-ence range in a manner that would flag these pa-tients could potentially help identify patients withborderline deficiency that could be addressedwithvitamin supplements.In conclusion, this study describes a simple

HPLC method with a suitable IS for evaluation ofboth vitamin B1 storage status and recent intake.This can provide an additional option to laborato-ries that are considering establishing their own as-say for vitamin B1 measurement, or improveworkflows for laboratories already performing an-other version of this assay. The use of a single sam-ple for both free thiamine and TDP measurementwill also eliminate an extra blood draw for patients,and potentially decrease the cost of testing.

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.

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