Short communication

Received 3 February 2011, Revised 10 March 2011, Accepted 14 March 2011 Published online in Wiley Online Library: 19 April 2011

(wileyonlinelibrary.com) DOI 10.1002/bmc.1636

On‐line solid‐phase extraction LC‐MS/MS forthe determination of Ac‐SDKP peptide inhuman plasma from hemodialysis patientsKoichi Inouea*, Ayaka Ikemuraa, Yoshinari Tsurutab,Kaname Tsutsumiuchic, Tomoaki Hinoa,d and Hisao Okaa,d

ABSTRACT: We developed a high‐throughput method based on on‐line solid‐phase extraction liquid chromatographytandem mass spectrometry (SPE‐LC‐MS/MS) to determine N‐terminal thymosin‐β fragment peptide (N‐acetyl‐seryl‐aspartyl‐lysyl‐proline, Ac‐SDKP) in human plasma samples. Quantification of Ac‐SDKP was performed using direct injection for on‐lineSPE based on C18, reversed‐phase LC separation and stable isotope dilution electrospray ionization‐MS/MS in multiplereaction‐monitoring (MRM) mode. The Ac‐SDKP‐13C6,

15N2 (m/z 496→137) was synthesized for the internal standard. TheMRM ion for Ac‐SDKP was m/z 488→129 (quantitative ion)/226. The limit of detection and lower limit of quantitation were0.05 and 0.1 ng/mL in standard solution, respectively. Recovery values were 98.3–100.4% with inter‐day (relative standarddeviation, RSD, 0.4–14.1%) and intra‐day (RSD, 0.8–19.7%) assays. This method was applied to the measurement of Ac‐SDKPlevels in plasma from hemodialyzed subjects. Concentrations were 0.59 ± 0.23ng/mL (pre‐hemodialyzed subjects, n=9) and0.44 ±0.19 ng/mL (post‐hemodialyzed subjects, n=9). All plasma Ac‐SDKP levels were decreased by dialysis. Thus, plasmaAc‐SDKP was decreased through dialysis in chronic kidney disease. The findings in this study will be useful for the treatmentof anemia in chronic kidney disease with dialysis. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (Ac‐SDKP); hemodialyzed subjects; liquid chromatography tandem mass spectrometry(LC‐MS/MS); on‐line solid‐phase extraction; human plasma

* Correspondence to: Koichi Inoue, Department of Physical and AnalyticalChemistry, School of Pharmacy, Kinjo Gakuin University, 2‐1723 Omori,Moriyama‐ku, Nagoya, Aichi 463‐8521, Japan. E‐mail: kinoue@kinjo‐u.ac.jp

a Department of Physical and Analytical Chemistry, School of Pharmacy,Kinjo Gakuin University, 2‐1723 Omori, Moriyama‐ku, Nagoya, Aichi463‐8521, Japan

b Meiyo Clinic, Meiyokai Medical Corporation, Japan

c College of Bioscience and Biotechnology, Chubu University, Japan

d Graduate School of Human Ecology, Human Ecology Major, Kinjo GakuinUniversity, Japan

Abbreviations used: Ac‐SDKP, N‐acetyl‐seryl‐aspartyl‐lysyl‐proline; FA,formic acid; MRM, multiple reaction monitoring.

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IntroductionN‐Acetyl‐seryl‐aspartyl‐lysyl‐proline (Ac‐SDKP) is a tetrapeptidefrom thymosin β4 by enzymatic N‐terminal cleavage, and ispresent in human blood. Thymosin β4 was proposed to be themetabolic precursor of Ac‐SDKP by prolyl oligopeptidase(Carvasin et al., 2004). Ac‐SDKP is a well‐known inhibitor ofhematopoietic stem cell proliferation (Guigon and Bonnet,1995). Moreover, high levels of expression of Ac‐SDKP occur inneoplastic diseases such as cancer (Liu et al., 2010). Otherreports have identified several active Ac‐SDKPs that blockinflammation and reduce fibrosis with unique functions (Sosneet al., 2010). Thus, Ac‐SDKP has recently been highlighted withregard to pathological angiogenesis.

There is little direct evidence of the role and state of Ac‐SDKP inhuman tissues. Early reports developed enzyme immunoassay(EIA) for the determination of Ac‐SDKP in human blood and cellsamples (Pradelles et al., 1990). Liozon et al. (1993) presentedresults demonstrating the presence of Ac‐SDKP in humans: serumlevels of 34 healthy subjects were found by EIA to be between 0.7and 2.5 pmol/mL (0.34–1.22 ng/mL), regardless of age and sex.Azizi et al. (1997) reported that the acute administration ofangiotensin‐converting enzyme inhibitor (ACEi) increases plasmaAc‐SDKP 5.5‐fold (ACEi group, 1.8 ng/mL; and non‐ACEi group,0.29 ng/mL). A few years later, Junot et al. (2001a) reported aselective EIA technique for a pharmacokinetic study in order tocompare similar Ac‐SDKP peptides. Many studies were performedblinded as to treatment using a commercially available andexpensive EIA kit for the clinical study (Azizi et al., 2006). On the

Biomed. Chromatogr. 2012; 26: 137–141 Copyright © 2011 John

other hand, these are the only two methods for measuringAc‐SDKP levels by a direct physicochemical analysis method suchas mass spectrometry (MS) (Junot et al., 2001b; Inoue et al., 2011).Recently, the prolyl endopeptidase (Prep)‐regulated peptides

have been of interest for peptidomics, and many patterns ofreleased small peptides have been discovered (Nolte et al., 2009).Prep would cleave thymosin β4, β10, β15A and other peptidesreleased to Ac‐SDKP analogs (peptides with similar structures suchas N‐acetyl‐alanyl‐aspartyl‐lysyl‐proline, Ac‐ADKP; and N‐acetyl‐seryl‐aspartyl‐lysyl‐proline‐NH2, Ac‐SDKP‐NH2; Hannappel, 2010).On the other hand, EIA methodology may give erroneous valuesdue to nonspecific binding to the antibody leading to an accurate

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estimation of the real concentration of Ac‐SDKP in human fluidssuch as blood, plasma and urine samples. For this reason, moreselective and accurate analytical techniques are needed toevaluate precise Ac‐SDKP levels based on the antigen–antibodyreaction. Thus, we developed stable isotope dilution liquidchromatography with tandem mass spectrometry (SID‐LC‐MS/MS)for the determination of Ac‐SDKP and minor Ac‐ADKP inhuman plasma (Inoue et al., 2011). SID‐LC‐MS/MS has goodsensitivity (limit of quantification, LOQ, 0.1 ng/mL) and accuratevalidation for monitoring the precise Ac‐SDKP levels in humanplasma. Unfortunately, SID‐LC‐MS/MS is not a high‐throughputmethod for large‐scale sampling from clinical and epidemiologicalstudies. Thus, the aim of this study was to develop a high‐throughput method based on on‐line solid‐phase extraction liquidchromatography tandem mass spectrometry (SPE‐LC‐MS/MS) forthe determination of Ac‐SDKP in human plasma. In addition, thismethodwas applied to themeasurement of plasmaAc‐SDKP levelsin pre/post hemodialysis patients.

Experimental

Chemicals and reagents

Synthetic Ac‐SDKP tetrapeptide (peptide purity, 98.8%; molecularweight, 487) was obtained from Peptide Institute Inc. (Osaka, Japan).HPLC‐grade water, methanol and formic acid (FA; 99%, LC/MS‐grade)were obtained from Wako Chemical Co. Inc. (Osaka). Purified waterwas obtained from a Milli‐Q purifying system (Millipore, Bedford, MA,USA). The pooled human plasma was obtained from Nissui Pharma-ceutical Co. (Tokyo, Japan). The Ac‐SDKP‐13C6,

15N2 was synthesizedbased on previous report (Inoue et al., 2011). This peptide was purifiedby HPLC fractionation and confirmed by ESI‐MS analysis.AcSDKP‐13C6,

15N2: ESI‐MS (H2O, positive mode); m/z 496 [M+H]+.The stock solutions (0.1mg/mL) for Ac‐SDKP and Ac‐SDKP‐13C6,

15N2

were prepared by dissolving the appropriate amount of standard inpure water. Calibration standards were prepared by diluting an aliquotof the stock solution in pure water.

We recruited subjects from November to December 2010 at MeiyoClinic, Toyohashi City in Aichi, Japan. This study was conducted with allsubjects’ written, informed consent and approved by the institutionalethical board for epidemiological studies. Blood were sampled from thepre/post‐hemodialyzed (n=9) subjects treated without any medicines,and stored at −30°C until use.

SPEcolumn

Drain

Direct injection of plasma sample

Pump A

Figure 1. On‐line SPE‐LC‐MS/MS system. Direct injection volume was 50μL o15–20min, solid line.

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SPE‐LC‐MS/MS analysis

The on‐line SPE‐LC‐MS/MS system is shown in Fig. 1. An LC pump (pump A)for on‐line SPEprocesswasperformedwith a Shimadzu LC‐20AD (ShimadzuCo., Kyoto, Japan) system. Liquid chromatography (pump B) was performedwith a Waters Alliance 2695 system (Waters Co., Milford, MA, USA). Theon‐line SPE column (TSK BSA‐ODS, 4.6×35mm: Tosoh, Co., Tokyo) wasconditioned using 0.1% FA in water with a flow rate of 0.5mL/min (pump A;Fig. 1). Separation was achieved on an Atlantis T3 column (2.1×150mm,3µm: Waters Co.) maintained at 30°C and the mobile phase consisted of0.1% FA in water (solvent A) and 0.1% FA in methanol (solvent B) usingpumpB (Fig. 1). The LC linear gradient was as follows: 2% solvent B at 0min,2%B at 4.0min, 40%B at 13min, 98%B at 13.1min, 98%B at 15min, and 2%B at 15.1minwith flow rate of 0.2mL/min. The switching valvewas changedat 5 min following the solid line (0‐5 min) to dotted line (5‐15 min) (Fig. 1).The injection volume was 50μL. The prepared and separated compoundswere detected with a Waters Micromass Quattro Premier triple quadrupolemass spectrometer (Waters Co.). Themass spectrometer was operated withan electrospray source in positive ionization mode. The ESI sourceconditions were: capillary voltage, 2.8 kV; extractor, 4 V; RF lens, 0 V; sourcetemperature, 110°C; and desolvation temperature, 400°C. The cone anddesolvation gas flowswere 50 and 450 L/h, respectively, and were obtainedfrom s nitrogen source (N2 Supplier Model 24 S, Anest Iwata Co., Yokohama,Japan). Argon was used as the collision gas and was regulated at 0.35mL/h;the multipliers were set to 650V. The LH resolution 1, HM resolution 1, ionenergy 1, LM resolution 2, HM resolution 2, and ion energy 2 were 12.0, 12.0,0.5, 12.0, 12.0 and 0.8, respectively.

Human plasma samples for SPE‐LC‐MS/MS

Plasma samples (100 μL) were added with internal solution (1 µg/mL;5 μL) as soon as possible after collecting blood in hospital. Then, thesesamples were stored at −30°C until use.

Analytical validation

In this study, the FDA guidance for industry (Bioanalytical MethodValidation) was modified for the analytical validation of endogenouspeptides in human plasma samples using stable isotope‐labeled internalstandard (IS) (FDA, 2001). Mostly, stable isotope‐labeled IS is used tocompensate for sample‐to‐sample differences occurring during on‐lineSPE preparation and LC‐MS/MS analysis such as absolute recovery,injection volume and matrix effects. Thus, calibration curves were usedwith the analyte‐to‐IS peak area ratios by weighted (1/x2) least‐squareslinear regression on consecutive days.

Separationcolumn

MS/MS

Pump B

f human plasma. Run time: 0–5min, solid line; 5–15min, dotted line; and

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SPE‐LC‐MS/MS for analysis of Ac‐SDKP

Plasma Ac‐SDKP levels were quantified by an eight‐point internalcalibration with IS. The calibration range was from 0.1 to 150ng/mL forstandard solutions. The acceptance criterion for a calibration curve was acorrelation coefficient (r2) of 0.99 or better. Spiked levels for qualitycontrol (QC) in pooled human plasma were selected of six replicates ofQC at three concentration levels (0.1, 5.0 and 150ng/mL). To determineintra‐day accuracy, replicate (n=6) analyses of plasma samples wereperformed on the same day. The inter‐day accuracy is expressed as therecovery and relative standard deviation (RSD, %), and was determinedtwice per day for three days. For the lower limit of quantification (LLOQ),the analyte response at the LLOQ was a based on signal‐to‐noise ratio(control) of 10. Process sample stability was evaluated with QC samples(0.5 and 10 ng/mL) after 24 h in the autosampler at 4°C. Bench‐topstability was evaluated for 6 h at room temperature. The freeze–thawstability was determined by comparing the freeze–thaw QC that hadbeen frozen and thawed three times at −30°C with normal QC samples(0.5 and 10 ng/mL). Long‐term stability was evaluated by analyzing QCsamples (0.5 and 10 ng/mL) stored at −30°C for 30 days. In addition, weevaluated the delivering and stored samples from hospital to laboratoryfor 24 h. Quality control samples (5.0 ng/mL of Ac‐SDKP in controlplasma samples) were spiked with IS in different states (state 1, inhospital after collecting blood from patients; and state 2, in thelaboratory after delivering samples for 24 h).

Results and discussion

On‐line SPE‐LC‐MS/MS analysis of Ac‐SDKP

Full‐scan and product ion spectra of the analytes wereinvestigated in infusion mode. They were detected underESI‐MS conditions based on the LC mobile phase. The protonatedmolecule of [M+H] + m/z 488 was detected in positive mode.When collision energy was used in product ions of [M+H] +, themajor fragment ions of m/z 488→129 (for quantification)/226were observed. Cone voltage of 37V and collision energy of27 eV in MS/MS condition were optimal conditions to achievehighly sensitive and selective multiple reaction monitoring(MRM) detection of Ac‐SDKP. The major fragment ions ofAc‐SDKP‐13C6,

15N2 at m/z 496→ 137 were observed. Quantita-tive analysis was performed using MRM mode in order tomaximize the sensitivity of the quantitative ion, and the ratioof analyte/IS.

Table 1. Analytical validation of plasma Ac‐SDKP levels using on

Stability Control levels (ng/mL) Spiked levels (n

Intra‐day assay 0.7 0.10.7 5.00.7 150.0

Inter‐day assay 0.7 0.10.7 5.00.7 150.0

Process/wet extracta 0.7 0.50.7 10.0

Bench‐topb 0.7 0.50.7 10.0

Freeze and thawc 0.7 0.50.7 10.0

Long‐termd 0.7 0.50.7 10.0

aAfter 24h in the autosampler at 4°C; bFor 6 h at roomtemperature; cA

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For the high‐throughput method based on on‐line SPE, thecolumn‐switching LC‐MS/MS system was developed to deter-mine Ac‐SDKP in human plasma samples (Fig. 1). Weinvestigated the sample volume and SPE columns for thissystem. The SPE columns of OASIS‐HLB (Waters Co.), TSKBSA‐ODS/S and BSA‐ODS (Tosoh Co.) were tested using 0.1%FA in water with a flow rate of 0.5mL/min using pump A. Thepeak’s sharpness and configuration on the BSA‐ODS were betterthan with the other columns. On the other hand, it was notpossible to obtain the sharp peak and configuration when alarger sample volume (>100 μL) was injected into the BSA‐ODScolumn. Thus, we selected an injection volume of 50 μL forBSA‐ODS. Standard solutions of Ac‐SDKP were prepared in purewater and added to a fixed concentration of Ac‐SDKP‐13C6,

15N2

for a calibration curve covering the concentration range from0.1 to 150 ng/mL for human plasma samples. Use of the methodto evaluate a measurement of endogenous Ac‐SDKP levels inhuman plasma samples was proposed. The matrix effect and thepossibility of ion suppression of specific Ac‐SDKP in biologicalsamples were observed by on‐line SPE preparation. The purposewas to examine whether on‐line SPE is useful for the traceanalysis of Ac‐SDKP in human plasma samples by MS/MSdetection. For this purpose, plasma samples were spiked at50 ng/mL, and were detected by on‐line SPE‐LC‐MS/MS. Thematrix effect values of the 50μL injection volumewere higher than95%. For the next step, on‐line extraction using the SPE columnwas performed according to the above‐described validation. Basedon these validations, on‐line SPE mode is sufficient for the recov-ery of Ac‐SDKP in human plasma and removing the high concen-trations of contaminating materials present in biological samples.

Analytical validations

The LLOQ in human plasma sample was detected as theconcentration of the lowest calibration by on‐line SPE‐LC‐MS/MS.The LLOQ was 0.1 ng/mL based on a signal‐to‐noise ration(plasma control sample) of 10. The results for intra‐day andinter‐day precision for Ac‐SDKP in QC samples are summarizedin Table 1. The intra‐day recovery and precision (RSD) were100.0–100.4 and 0.4–14.1%, respectively. The inter‐day recovery

‐line SPE‐LC‐MS/MS

g/mL) Detection levels (ng/mL) Recovery [RSD] %

0.8 100.0 [14.1]5.7 100.4 [3.6]

151.1 100.2 [0.4]0.8 98.3 [19.7]5.7 100.1 [0.9]

150.3 99.7 [0.8]1.2 98.0 [2.9]

10.7 100.3 [0.8]1.2 103.0 [3.2]

10.6 99.4 [1.3]1.2 105.7 [1.4]

10.7 100.4 [0.9]1.2 104.7 [2.6]

10.7 100.4 [0.9]

fter three freeze–thawcycles at−30°C; dFor 30 days at−30°C;n=6.

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and precision (RSD) were 98.3–100.1 and 0.8–19.7%, respective-ly. The results of stability studies are shown in Table 1. Qualitycontrol samples were subjected to long‐term storage (−30°C),and to freeze–thaw stability studies. All stability tests wereconducted at two concentration levels (0.5 and 10.0 ng/mL) withsix determinations for each. The bench‐top stability results(103.0 and 99.4%; RSD 3.2 and 1.3%) indicated that Ac‐SDKP inhuman plasma is stable for at least for 6 h at room temperature.Using this result, the preparation of human plasma should becompleted within 6 h at room temperature. Long‐term stabilityresults (104.7 and 100.4%; RSD 1.4 and 0.9%) indicated that theAc‐SDKP level was stable for 30 days at −30°C. Thus, the stabilityof Ac‐SDKP at −30°C was used for the storage time (30 days)until analysis. Figure 2 shows the MRM chromatograms ofAc‐SDKP and IS in human plasma for the recovery test, and in a real

(min)

488 > 128.71.53e4

14.6

496 > 136.81.63e5

14.6

Time0 2 4 6 8 10 12 14 16

488 > 128.73.44e3

14.6

496 > 136.81.15e5

14.6

Rel

ativ

e re

spon

seR

elat

ive

resp

onse

Rel

ativ

e re

spon

seR

elat

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resp

onse

Ac-SDKP

Ac-SDKP-13C6, 15N2

Ac-SDKP

Ac-SDKP-13C6, 15N2

(A)

(B)

(C)

(D)

Figure 2. MRM chromatograms of Ac‐SDKP and IS in human plasma forrecovery test and real sample. Recovery test: 5.0 ng/mL of Ac‐SDKP inhuman plasma. (A) Recovery test: IS in human plasma. (B) Clinical study:0.59 ng/mL of Ac‐SDKP in hemodialyzed subject. (C) Clinical study: IS inhemodialyzed subject.

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human plasma sample. The choice of the IS is a critical aspect ofquantitative methods because it influences precision andaccuracy. In addition, we investigated the validation for deliv-ering samples between hospital and analytical laboratory for24 h. The recoveries were 98.0% (RSD 2.7%) in state 1, and80.2% (RSD 8.4%) in state 2. This indicated that state 1 (spikedwith IS in hospital) is better than state 2 (spiked with IS inlaboratory). Therefore, in this experimental trial, we adoptedstate 1 for the clinical application of measuring Ac‐SDKP inplasma from pre/post‐hemodialyzed subjects. Based on thesevalidation data, this reliability of this analytical procedure wasuseful for monitoring Ac‐SDKP levels in various human plasmasamples.

Clinical application of Pre/post‐hemodialyzed subjects

The role of erythropoiesis‐stimulating agents (ESAs) in treatingthe anemia of chronic kidney disease has been evaluated invarious clinical reports (Johansen et al., 2010). On the otherhand, inhibitory hematopoiesis of Ac‐SDKP is indicated inchronic kidney disease (Guigon and Bonnet, 1995). Thus, it isvery important for Ac‐SDKP levels to be monitored in chronickidney disease when treating anemia using ESAs. A previousstudy reported that levels of Ac‐SDKP in anemic, nonanemic andhealthy control subjects ranged from 0.5 to 4.5 nM (0.24 to2.2 ng/mL; van der Meer et al., 2005). Le Meur et al. (2001)reported that a high Ac‐SDKP level acts as a uremic toxin,causing partial resistance to erythropoietin and inhibitingerythropoiesis. It is necessary to enhance Ac‐SDKP level inchronic kidney disease, such as in hemodialyzed patients.

Plasma Ac‐SDKP levels were significantly greater in hemodia-lyzed subjects, 10.3 ± 3.9 pmol/mL (5.0 ± 1.9 ng/mL), than incontrols, 1.8 ± 0.2 pmol/mL (0.9 ± 0.1 ng/mL) (Le Meur et al.,2001). Recently, we reported that plasma Ac‐SDKP levels were0.4 ± 0.2 ng/mL (healthy subjects, n= 7) and 0.6 ± 0.2 ng/mL(hemodialyzed subjects, n= 34), respectively (Inoue et al.,2011). We explained the differences in Ac‐SDKP levels in patientsrelieved by dialysis. Figure 3 shows the Ac‐SDKP levels before/after dialysis. Plasma Ac‐SDKP levels were 0.59 ± 0.23 ng/mL

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7 8 9

Before

After

Plas

ma

Ac-

SDK

P le

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(ng

/mL

)

Hemodialysis patients

Figure 3. Ac‐SDKP levels in pre/post hemodialyzed subjects (n=9).Detected levels: plasma Ac‐SDKP levels were 0.59± 0.23 ng/mL (pre‐hemodialyzed subjects, n=9), and 0.44± 0.19 ng/mL (post‐hemodialyzedsubjects, n=9).

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SPE‐LC‐MS/MS for analysis of Ac‐SDKP

(pre‐hemodialyzed subjects, n = 9) and 0.44 ± 0.19 ng/mL(post‐hemodialyzed subjects, n= 9), respectively. In addition,all plasma Ac‐SDKP levels were decreased by dialysis. Thus,the plasma Ac‐SDKP level was reduced through dialysis inchronic kidney disease. This finding is useful in the treat-ment of anemia using ESAs in chronic kidney disease withdialysis.

In this data, for the first time Ac‐SDKP levels in pre/post‐dialyzed subjects were compared using a reliable on‐lineSPE‐LC‐MS/MS method. This high‐throughput analyticalmethod would be useful for the quantitative determinationof plasma Ac‐SDKP levels in large‐scale studies.

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