Anal Bioanal Chem DOI 10.1007/s00216-013-7540-7

RESEARCH PAPER

Conditions for sample preparation and quantitative HPLC/MS-MS analysis of bulky adducts to serum albumin with diolepoxides of polycyclic aromatic hydrocarbons as models Emelie Westberg & Ulla Hedebrant & Johanna Haglund & Tomas Alsberg & Johan Eriksson & Albrecht Seidel & Margareta Törnqvist

Received: 11 July 2013 / Revised: 18 November 2013 / Accepted: 28 November 2013 # Springer-Verlag Berlin Heidelberg 2014

Electronic supplementary material The online version of this article (doi:10.1007/s00216-013-7540-7) contains supplementary material, which is available to authorized users.

His-Pro, PAHDE-His-Pro-Tyr and PAHDE-Lys. Alkaline hydrolysis under optimised conditions gave the BPDE-His as the single analyte of alkylated His, but also indicated degradation of this adduct. It was not possible to obtain the BPDE-His as one analyte from BPDE-alkylated SA through modifications of the enzymatic hydrolysis. The BPDE-His adduct was shown to be stable during the weak acidic conditions used in the isolation of SA. Enrichment by HPLC or SPE, but not butanol extraction, gave good recovery, using Protein LoBind tubes. A simple internal standard (IS) approach using SA modified with other PAHDE as IS was shown to be applicable. A robust analytical procedure based on digestion with pronase, enrichment by HPLC or SPE, and analysis with HPLC/MS-MS electrospray ionisation was achieved. A good reproducibility (coefficient of variation (CV) 11 %) was obtained, and the achieved limit of detection for the studied PAHDE, using standard instrumentation, was approximately 1 fmol adduct/mg SA analysing extract from 5 mg SA.

E. Westberg : U. Hedebrant : J. Haglund : J. Eriksson : M. Törnqvist (*) Division of Environmental Chemistry, Department of Materials and Environmental Chemistry, Stockholm University, Svante Arrhenius väg 16c, 10691 Stockholm, Sweden e-mail: [email protected]

Keywords Bulky serum albumin adducts . Polycyclic aromatic hydrocarbons . Extraction (SPE | HPLC | butanol) . Diol epoxides . Mass Spectrometry . Hydrolysis (pronase enzymatic | alkaline)

T. Alsberg Department of Applied Environmental Science, Stockholm University, Svante Arrhenius väg 8, 11418 Stockholm, Sweden

Introduction

Abstract Stable adducts to serum albumin (SA) from electrophilic and genotoxic compounds/metabolites can be used as biomarkers for quantification of the corresponding in vivo dose. In the present study, conditions for specific analysis of stable adducts to SA formed from carcinogenic polycyclic aromatic hydrocarbons (PAH) were evaluated in order to achieve a sensitive and reproducible quantitative method. Bulky adducts from diolepoxides (DE) of PAH, primarily DE of b enzo [a ]pyr ene (BPDE) and also DE of dibenzo[a ,l ]pyrene (DBPDE) and dibenzo[a ,h ]anthracene (DBADE), were used as model compounds. The alkylated peptides obtained after enzymatic hydrolysis of human SA modified with the different PAHDE were principally PAHDEEmelie Westberg and Ulla Hedebrant contributed equally to this work.

A. Seidel Biochemical Institute for Environmental Carcinogens, Prof. Dr. Gernot Grimmer-Foundation, Lurup 4, 22927 Grosshansdorf, Germany Present Address: J. Haglund MetaSafe AB, Biovation Park Telge, 15136 Södertälje, Sweden

In work aiming at cancer risk assessment of exposure to genotoxic compounds, it is important to be able to measure their effective dose in vivo. In general, genotoxic compounds possess either an electrophilic reactivity themselves or are metabolically converted to such reactive intermediates which can subsequently form adducts with biomacromolecules.

E. Westberg et al.

Adducts to haemoglobin (Hb) or serum albumin (SA) offer possibilities for in vivo measurement of short-lived electrophiles and have been used in biomonitoring studies to detect and quantify exposures to genotoxic agents in humans [1, 2]. These are primarily adducts from low molecular weight compounds, as simple epoxides [3] and benzene metabolites [4], as well as aromatic amines [5], which have been monitored by gas chromatography–mass spectrometry (GC/MS) with high sensitivity. Biomonitoring of protein adducts from high molecular weight compounds, for instance, adducts from aflatoxin to SA [6, 7], has mainly relied on immunoassays. There are several requirements to be fulfilled to make an analysis of protein adducts a useful method for quantification of in vivo doses (area under the time–concentration curve) of the electrophile. The method should be sufficiently sensitive for application to exposed humans, and it should give structure-specific information, which means that the analysis should be performed by MS methods. Preferably, the analyte should include a component (“tag”) from the protein implying that it originates from a covalently bound adduct, to eliminate the risk that, for instance, hydrolysis products of the electrophile are measured as in vivo formed adducts. Further, the adducts should be stable in vivo, and the method be applicable also to experimental animals to facilitate in vivo dose calculations and species extrapolations of exposure-in vivo dose relationships. For the well-studied polycyclic aromatic hydrocarbons (PAH), there is no such method available fulfilling the requirements for accurate in vivo dosimetry of the genotoxic metabolites. Several PAH are classified as carcinogenic in experimental animals and benzo[a]pyrene (BP) is classified as a human carcinogen [8]. The general population is exposed to PAH through urban air pollution, originating from combustion of fossil fuels and biomass, and through intake of contaminated foods, particularly grilled and smoked food [9]. BP is often used as a marker of PAH exposure, it is extensively studied and its metabolic transformation is well characterised [10]. BP is metabolised to diolepoxides (DE), (±)-syn - and (±)-anti -BP-7,8-diol-9,10-epoxides (syn - and anti -BPDE), of which the predominant isomer (+)-anti BPDE has shown the highest tumour-inducing activity [11, 12]. BPDE has been demonstrated to form carboxylic ester adducts in Hb and SA with aspartate (Asp), glutamate (Glu) or C-termini [13–16]. The ester adducts are susceptible to hydrolysis in vitro and also in vivo and are, thus, not perfect for in vivo dosimetry. Stable adducts from BPDE to histidine (His) has, however, been identified in vitro in both human Hb [17] and human SA [18]. The reaction site in human SA was identified as His146 [18], and it has been reported that this His adduct was formed from the (-)-anti-BPDE [13]. A comparison of human SA and human Hb alkylated in vitro with (±)-anti-BPDE, showed that 70 and 10 %, respectively, of the total BPDE products consisted of adducts to His [17].

Analysis of stable His adducts to SA thus would be the method of choice for in vivo dosimetry of BPDE. Adducts to both SA and Hb, with BP as a marker substance for PAH exposure, have been studied with different methods (reviewed by Kaefferlein et al. [19]). In a number of human studies, BPDE adducts formed with SA have been measured by an enzyme-linked immunosorbent assay (ELISA) in peptides after enzymatic digestion or as tetrols after their release through hydrolysis. Generally, the ELISA method could be interpreted as a measure of PAH exposure, as there is crossreactivity of the used antibody with other PAH [19]. By sensitive chemical-specific methods (based on detection with MS or fluorescence), adducts from (+)-anti-BPDE to SA and Hb have been analysed as tetrols after mild hydrolytic release. One difficulty with this method is that noncovalently bound BP tetrols must be removed completely before an accurate determination of the level of covalently bound adducts (cf. [16]). Kaefferlein et al. [19] conclude that despite the large number of human studies published, the overall data on protein adducts from BP in humans are inconclusive and that development of a sensitive and specific method for the measurement of BP protein adducts is required for biomonitoring studies. The above conclusion highlights the need for a new analytical method for PAH adducts to blood proteins, which also fulfils the above stated requirements of a method for in vivo dosimetry. We earlier initiated such development and reported on an analysis of stable adducts from (±)-anti-BPDE to His [17] based on cleavage of BPDE-alkylated SA with hydrazine and subsequent high-performance liquid chromatography (HPLC)/MS quantification. Despite promising results, this development was abandoned due to the inconvenience of handling hydrazine. There is only one published in vivo study on BPDE adducts to His in SA, in which BPDE-histidineproline (BPDE-His-Pro) was detected in human SA after enzymatic digestion by using HPLC combined with highly sensitive laser-induced flurorescence (LIF) spectroscopy [20]. An overall aim is to broaden in vivo dosimetry by using MS techniques to measure protein adducts from high molecular weight electrophilic compounds. The general objective of the present study was to evaluate conditions for isolation, enrichment and quantitative analysis by liquid chromatography tandem mass spectrometry (HPLC/MS-MS) of chemically stable bulky adducts to SA. The specific objective was to identify adducts and target analytes of the PAH used as models, i.e., the diolepoxide metabolites of BP, dibenzo[a ,h ]anthracene (DBA) and dibenzo[a ,l ]pyrene (DBP1) (Fig. 1), and to determine which limit of detection (LOD) could be obtained by applying the optimised conditions for analysis using standard MS instruments. 1 Also designated as dibenzo[def ,p ]chrysene—the most systematic IUPAC name is naphtho[1,2,3,4-pqr]tetraphene.

Analysis of bulky adducts to serum albumin with PAH diolepoxides

Fig. 1 Structures of the studied diolepoxides of polycyclic aromatic hydrocarbons; anti -BP-7,8-diol-9,10-epoxide (BPDE ), anti dibenzo[a ,h ]anthracene-3,4-diol-1,2-epoxide (DBADE ) and anti dibenzo[a,l]pyrene-11,12-diol-13,14-epoxide (DBPDE). Absolute stereochemistry is not implied

ratio). After incubation for 1 h at 37 °C, the pH was adjusted to 4 using HCl (1.0 M). Cold (∼6 °C) saturated ammonium sulfate (10 mL) was slowly added during stirring, and the BPDE-alkylated SA (BPDE-SA) was precipitated (20 min). The sample was centrifuged at 4,500×g for 10 min, and the precipitate, i.e. BPDE-SA, was washed with 20 mL MeOH, followed by 2×20 mL ethyl acetate and finally with 10 mL pentane. After each wash, the sample was centrifuged at 1,600×g for 10 min. The final precipitate was left to dry overnight at room temperature.

Materials Enzymatic hydrolysis of BPDE-SA The following chemicals were used: Human SA crystallised and lyophilised (A9511) and sheep SA lyophilised (A3264) from Sigma (Steinheim, Germany); BPDE from the National Cancer Institute, Chemical Carcinogen Reference Standard R e p o s i t o r y ( K a n s a s C i t y, K S , U S A ) ; ( ± ) - a n t i dibenzo[a,h]anthracene-3,4-diol-1,2-epoxide (DBADE); and (±)-anti -dibenzo[a ,l ]pyrene-11,12-diol-13,14-epoxide (DBPDE) were synthesised as described earlier [21, 22]. Pronase of 7 U/mg (165921) and papain of 30 U/mg (108014) were from Roche Diagnostics Scand AB (Mannheim, Germany); RapiGest™ (sodium 3-[(2-methyl-2undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate) were from Waters Corporation (Milford, MA, USA); and methanol (MeOH) and water were from BDH (Poole, England). All other chemicals and solvents used were of analytical grade. Protein LoBind® tubes (Eppendorf, Hamburg, Germany) were used in the final experimental procedures. Caution BPDE, DBADE and DBPDE are mutagens and carcinogenic in animal cancer tests and should be handled with care, following appropriate safety procedures.

BPDE-SA (10 mg) was dissolved in 350 μL of ammonium bicarbonate (NH4HCO3, 50 mM), and an internal standard (see below) dissolved in NH4HCO3 (50 mM, 50 μL) was added. Then 100 μL of pronase (9 mg/mL), dissolved in NH4HCO3 (50 mM), was added. The solution was incubated at 37 °C overnight (∼20 h). For enrichment of adducts from the enzymatic digest of BPDE-SA, isolation by HPLC-UV or solid phase extraction (SPE) was used (see below). Isolation and off-line enrichment of BPDE adducts HPLC-UV detection The enzymatic digest of BPDE-SA was fractionated and analysed by semi-preparative HPLC-UV. An HPLC (LKB, Bromma, Sweden), with a UV detector (Shimadzu, Kyoto, Japan) set at 345 nm and a Kromasil C18 column (5 μm, 10×250 mm; Hichrom, Reading, UK) was used. Eluent A was water/MeOH (95:5, v /v ), and eluent B was water/MeOH (5:95, v/v), with 0.1 % formic acid in both A and B. A linear gradient from 5 to 100 % B in 30 min and then 100 % B for 10 min was used. The flow rate was 3 mL/min. Acquisition and processing of data was performed using ELDS win 1.1 (Chromatography Data Systems, Svartsjö, Sweden). Fractions containing the analytes were collected and concentrated to 100 μL using a vacuum centrifuge.

Experimental In this section, the final procedures are given, which were settled after evaluation of conditions for isolation of PAHDEmodified SA, enrichment, identification and quantification of the analytes, as shortly described in Table 1 in section “Tests to improve the sensitivity of the analytical method”. Preparation of BPDE-alkylated SA The standard used for the development work was generated by in vitro incubation of human SA with BPDE. Pure human SA (405 mg) was dissolved in 5 mL H2O, and the pH was adjusted to 7.5 with NaOH (0.1 M, approx. 100 μL). A suspension of 100 μL of BPDE (10 μg/μL) in tetrahydrofuran was added to the solution of SA (SA/BPDE 1:0.45 molar

SPE Isolation of adducts after enzymatic cleavage of SA was alternatively done by SPE using a Sep-Pak Plus, C18 Column (360 mg) from Waters (Milford, MA, USA). The SPE column was conditioned with 5 mL MeOH and equilibrated with 5 mL H2O, and the enzymatic digest from 10 mg BPDE-SA, dissolved in 1 mL H2O, was added to the column. The column was washed with 5 mL sodium acetate (0.5 M, pH 5.0), followed by 5 mL H2O and finally with 5 mL MeOH (30 %). The sample was eluted with 2 mL MeOH (95 %) and concentrated to 90 μL using a vacuum centrifuge. Identification and quantification Analysis of adducts by HPLC/MS-MS The fractions from either preparation technique, HPLC or SPE, were analysed

E. Westberg et al. Table 1 Conditions for parameters evaluated in the procedures for analysis of adducts to SA from bulky molecules, using BPDE adducts as models

Procedure studied

Evaluated parameters

Conditions tested

Adhesion to material

Tube material

Precipitation of SA

pH Time in acidic solution Concentration of NH4HCO3 to dissolve SA Type of enzyme addition

Eppendorf LoBind®, polypropylene, unsilanised glass, silanised glass pH 4 and pH 7 0.3, 20 and 40 h 50 and 200 mM

Hydrolysis

Enzymatic hydrolysis

Alkaline hydrolysis

Enrichment of adducts HPLC/MS-MS parameters

Matrix effect in the HPLC/MS-MS analysis

using HPLC/MS-MS, and adducts were identified as described below. An HPLC (LC-20 Prominence System, Shimadzu, Kyoto, Japan) coupled to a triple quadrupole MS (API 3200 Q TRAP, Applied Biosystems/MDS Sciex, Toronto, Canada) was used. The HPLC column was a C18 column with 5-μm particles (Ace, 1.0×150 mm). Eluent A was water/ MeOH (95:5, v/v), and eluent B was water/MeOH (5:95, v/v). Formic acid (0.1 %) was added to both eluents A and B. The eluent was 0 % B for 2 min, followed by a linear gradient from 0 to 100 % in 18 min followed by 100 % B for 10 min. The flow rate was 50 μL/min. The MS was operated with electrospray ionisation in the positive ion mode (ESI+). The scan modes were full scan, product ion scan (PIS) and multiple reaction monitoring (MRM). Unit mass resolution was used for all analyses, and the data were acquired in the centroid mode. In the MRM mode, the ion source temperature was 200 °C, ion spray voltage was 5,500 V, curtain gas 17, collision gas 5, GS1nebulising gas 30 and GS2-auxiliary gas 30 (the latter four units are arbitrary from the Analyst software and the gases used were N2). MS parameters optimised for each analyte and their respective ranges were as follows: declustering potential 40– 60 V, entrance potential 4–10 V and collision energy 15– 60 V. Acquisition and processing of data were performed using the Analyst software version 1.4.1 (Editions Applied Biosystems/MDS Sciex Instruments, Toronto, Canada). Two MRM transitions were used for most of the analytes. Quantification of SA and BPDE adducts with HPLC-UV For determination of the albumin concentration in the in vitro

Enzyme/substrate Concentration of SA Final concentration of NaOH Temperature; time Method Mobile phases and electrospray polarity Amount SA/sample

Pronase; pronase + papain; pronase + RapiGest 0.1 and 0.4 mg pronase/mg SA 0.2 and 20 mg SA/mL 1, 2 and 4 M 23, 100 and 130 °C; 0.5–46 h SPE, HPLC, butanol extraction pH 3 and pH 7 MeOH and AcN; formic acid and trifluoroacetic acid; positive and negative 10, 25 and 40 mg

alkylated SA sample, the absorbance of BPDE-SA was measured at 280 nm with a Hitachi U-3000 spectrophotometer (Tokyo, Japan). A linear calibration curve was obtained with pure human SA and one concentration (5 mg/mL, 50 mM NH4HCO3) was then used for one-point calibration. To quantify the BPDE adduct levels, a fraction of the in vitro alkylated SA was hydrolysed with pronase and analysed by HPLC-UV as described above. Since synthetic standard(s) for quantification of BPDE adduct to His was not available, the adduct level of the in vitro BPDE-alkylated SA used as standard was quantified from HPLC-UV analysis using Eq. 1. C inj ¼

A⋅f Q⋅ε⋅l⋅V inj

ð1Þ

where C inj = concentration in the injected sample (moles per litre); A = peak area (microvolt second); f = flow rate (litres per second); Q = output voltage/absorption unit (microvolt per AU); ε = extinction coefficient (per molars per centimetre); l = cuvette length (centimetres); and Vinj = injected volume (litres). The instrument-specific Q value (in Eq. 1) for the HPLCUV used was calculated from analysis of a substance with known extinction coefficient in known concentration. The extinction coefficient (ε ) for BPDE-deoxyguanosine ε 345 nm =2.9×104 M−1 cm−1 [23] was used as an approximate ε to estimate the BPDE adduct levels (to His and Lys) in SA. The concentration of the studied BPDE adducts (picomoles of

Analysis of bulky adducts to serum albumin with PAH diolepoxides

BPDE adduct per milligram of SA) was calculated from the HPLC peak area according to Eq. 1. The adduct concentration of the BPDE-SA was estimated through several HPLC-UV analyses at different concentrations.

calibration sample to check that there was no carryover. Double injections of 40 respective 50 μL, which corresponded to 4 and 5 mg SA, from each calibration point were analysed by HPLC/MS-MS in the ESI+ mode using MRM.

Identification and quantification of adducts to SA from DBADE and DBPDE After evaluation of the method for analysis of BPDE adducts, the procedures were used to identify adducts to SA from DBADE and DBPDE. Adducts were generated by incubation of these compounds with SA (reactions were done as described for BPDE, but reaction time was adjusted to 20 h). The samples of alkylated SA were digested using pronase and enriched and analysed by HPLC/MS-MS, as described above for BPDE-SA. The level of the adducts from DBADE and DBPDE in in vitro alkylated SA were estimated by calculations using the HPLC-UV areas of the analyte peaks from digested and fractionated SA as described above for BPDE-SA. The extinction coefficients used for DBADE was ε 2 91 n m = 9.25 × 104 M −1 cm−1 (personal communication B. Jernström, Karolinska Institute, Stockholm, Sweden) and for DBPDE (MeOH) ε 294 nm =4.23×104 M−1 cm−1 [24].

Tests to improve the sensitivity of the analytical method To improve the recovery and LOD in the analysis of SA adducts, different steps/conditions and materials used in the analytical procedure were evaluated (Table 1). These were tube materials in the work-up process, influence of time and pH in the precipitation step, alkaline and variations of enzymatic hydrolysis, as well as different procedures for enrichment (Electronic Supplementary Material Fig. S1) and the amount of sample injected (Electronic Supplementary Material Fig. S2) for analysis of the studied adducts. For detailed information about the evaluations and Fig. S1 and S2, see the Electronic Supplementary Material. In the evaluation process, the produced standard of in vitro BPDEalkylated SA was used, and the different parameters were compared in parallel experiments.

Results Internal standard for quantification in the HPLC/MS-MS analysis The generation of internal standards (IS) for HPLC/MS-MS quantification required an approach which possibly could be generalised. The IS should be useful for several adducts from PAHDE and also should adjust for recovery in the enzymatic digestion, as well as for MS response. As a way to generate a standard for quantification in a convenient way (and reduced cost), it was tested whether SA alkylated with a PAHDE other than the one under study could be used. Thus, DBPDE-alkylated human SA was evaluated as internal standard for BPDE adducts, and BPDE-alkylated human SA was tested as internal standard for DBPDE- and DBADE-alkylated human SA. The IS was added to the samples before enzymatic hydrolysis. Estimation of LOD and reproducibility for analysis of adducts with HPLC/MS-MS After ezymatic hydrolysis of in vitro alkylated SA, the PAHDE adducts were observed as PAHDE-His-Pro (dipeptide), PAHDE-His-Pro-Tyr (tripeptide) and PAHDE-Lys (see “Results”). The LOD, defined as three times the signal-to-noise (S/N) ratio, in the HPLC/MS-MS analysis was obtained from matrix-matched calibration samples. The summed areas of the di- and tripeptide peaks were used to calculate the adduct levels. To obtain calibration samples, different amounts of PAHDEmodified SA were added to unmodified SA, to a total amount of 10 mg SA. In the final work, sheep SA was used as matrix instead of human SA. Solvent was injected between every

Identification and quantification of PAHDE adducts formed with SA HPLC-UV chromatograms from the analysis of digests of BPDE-SA are shown in Fig. 2. The peaks from BPDE-SA, hydrolysed with pronase (Fig. 2a), were identified with HPLC/MS-MS as BPDE-Lys (peak 1), BPDE-His-Pro (peak 2), BPDE-His-Pro-Tyr (peak 4) and BPDE tetrols (peak 5 and peak 6). After alkaline hydrolysis of BPDE-SA (Fig. 2b), the only analyte of the His adduct identified was the modified amino acid, BPDE-His (peak 3). BPDE-Lys (peak 1) and BPDE tetrols (peak 5 and peak 6) were also present. The structural elucidation of the analytes was based on their diagnostic fragmentation pattern (Fig. 3). For BPDE, the His adduct analytes (as di- and tripeptide) give the largest peaks in the analysis by HPLC/MS-MS (after pronase hydrolysis). BPDE-HisPro, -His-Pro-Tyr and -Lys were quantified to 270, 380 and 20 pmol adduct/mg SA, respectively (from incubation 1 h, initial concentration 0.66 mM BPDE). The adduct levels were calculated from the HPLC-UV (λ 345 nm) areas using Eq. 1. The analysis of the DBADE-SA and DBPDE-SA after hydrolysis with pronase showed alkylation of the same amino acids and the analytes correspond to those found for BPDE, except that DBPDE-His-Pro-Tyr was not observed. The fragmentation patterns from the DBADE and DBPDE adducts

E. Westberg et al.

5

a UV-Absorbance, 345nm

Fig. 2 HPLC-UV chromatogram from analysis of SA alkylated with BPDE. Trace a after hydrolysis with pronase, and trace b after alkaline hydrolysis. Fractions of the eluent from the different peaks were collected and analysed using HPLC/MS-MS. Peak 1 was identified as BPDELys, peak 2 as BPDE-His-Pro, peak 3 as BPDE-His, peak 4 as BPDE-His-Pro-Tyr, and peaks 5 and 6 as BPDE tetrols

4

2

6

1

Time (min.)

5

UV-Absorbance, 345nm

b 3 6 1

Time (min.) were also in agreement with the fragmentation of the BPDE adducts (Table 2). For DBADE and DBPDE, the lysine adduct

showed the largest peak. The level of DBADE-His-Pro, -HisPro-Tyr and -Lys was quantified to 170, 110 and 270 pmol adduct/mg SA, respectively (λ 291 nm). For the DBPDE-SA, the level of DBPDE-His-Pro was quantified to 50 pmol/mg and DBPDE-Lys to 134 pmol/mg (λ 294 nm). Development of the analytical procedure A summary of the results achieved from the evaluated procedures briefly described in Table 1 are summarised in Table 3. Internal standard, quantification and reproducibility

Fig. 3 Fragmentation of BPDE-His adducts from positive electrospray ionisation (ESI+)

From calibration samples of BPDE-SA adducts, prepared many times during the development to evaluate LOD etc., linear calibration curves in the HPLC/MS-MS analysis were obtained with good repeatability (number of calibration points were always ≥8). No carryover was observed between the runs. The use of SA alkylated with another PAHDE than the target compound, added to matrix-based calibration samples before enzymatic digestion, was evaluated as IS for the quantitative analysis. The correlation coefficients, r 2, increased with the use of this IS approach (0.99 compared to 0.92 at medium levels in the calibration curves, at higher levels, the

Analysis of bulky adducts to serum albumin with PAH diolepoxides Table 2 Precursor and product ions from HPLC/MS-MS (ESI+) MRM analysis of adducts formed in SA from the diolepoxides of the three studied PAH and obtained after hydrolysis by pronase of SA

Analyte BPDE-His-Pro DBADE-His-Pro DBPDE-His-Pro BPDE-His-Pro-Tyr DBADE-His-Pro-Tyr DBPDE-His-Pro-Tyr BPDE-Lys DBADE-Lys DBPDE-Lys

[M+H]+ 555 581 605 [M+H+] 718 744 n.d. (768) [M+H+] 449 475 499

difference was not that evident). A calibration curve for BPDE-His based on the summed peak areas of BPDE-HisPro and BPDE-His-Pro-Tyr with DBPDE-Lys as IS is shown in Fig. 4. The calibration curve was linear from ca 0.03 pmol/ mg SA for 4 orders of magnitude tested (whole range 0.0007–

[His-Pro]+ 253 253 253 [His-Pro-Tyr]+ 416 416 n.d. [Lysine]+ n.d. 147 147

[diol-CO]+ 257 283 307 [diol-CO]+ 257 283 n.d. [diol-CO]+ 257 283 307

[triol]+ 303 n.d. 353 [triol]+ 303 n.d. n.d. [triol]+ 303 n.d. 353

[diol]+ 285 311 335 [diol]+ 285 311 n.d. [diol]+ n.d. 311 335

200 pmol adducts/mg SA). The r 2 values decreased when ranges of adduct levels were lowered, e.g. 0.001–56 pmol/mg, r 2 =1.000 (n =10); 0.001–0.25 pmol/mg, r 2 =0.99 (n =6); and 0.001–0.08 pmol/mg, r 2 =0.92 (n =5). The calibration curves of the adduct levels for the other PAHDE were linear with

Table 3 Results from the evaluation of parameters in the method for analysis of adducts formed after reaction between serum albumin and diolepoxides of PAH Procedure studied

Results and comments of evaluated parameters

Adhesion to material Tube material

Eppendorf LoBind®

Precipitation of SA

pH

pH 4

Time in acidic solution

0.3 h

Concentration of NH4HCO3 to dissolve SA Type of hydrolysis

50 mM

Hydrolysis

Enzymatic hydrolysis Enzyme/substrate

Enzymatic with pronase

0.1 mg pronase/mg SA

Concentration of SA

20 mg SA/mL

Final conc. of NaOH

2M

Temperature; time

100 °C, 5–24 h

Enrichment of adducts

Method

SPE or HPLC

HPLC/MS-MS parameters

Composition of mobile phases and electrospray charge

MeOH with formic acid in positive mode

Matrix effect in the HPLC/MS-MS

Response in the MS analysis

10 mg

Alkaline hydrolysis

Increased yield with Eppendorf LoBind® tubes (>40%), no large difference between the other materials The adducts were stable and the combined yield of BPDE-His-Pro and -His-Pro-Tyr was the same at both pH studied, although a shift of the adduct ratio was seen Decreasing amount of SA/mg precipitate was seen with increasing time but the same amount adduct/mg SA. Probably more salt is formed over time An increase in yield of ca 25 % the yield with 50 mM Use of pronase alone gave lowest LOD (12 times lower compared to alkaline hydrolysis) even though two analytes had to be considered. Addition of RapiGest or papain had no effect No obvious effect was observed on the yield by the increased enzyme–substrate ratio tested No obvious effect was observed on the yield by the decreased concentration of SA Stronger conc. of NaOH resulted in degradation of the adduct Lower temperature gave low yields irrespective of time. The time for hydrolysis was not crucial SPE and HPLC were comparable methods regarding the yield but SPE preferable due to less time and solvent consumption. Butanol extraction gave 307.1 Da

2000

10.00 5.00 0.00 HPLC (n=6)

SPE 1 (n=6)

SPE 2 (n=6)

Fig. 5 Comparison of the repeatability of the final method for HPLC/MS-MS measurements of BPDE-His in SA after enrichment by HPLC or SPE (duplicate samples and three concentrations, 0.2–2.1 pmol BPDE-His-Pro and BPDE-His-Pro-Tyr adduct/mg SA). The bars show the peak area ratio (combined BPDE-His modified di- and tripeptides divided by the internal standard DBPDE-Lys) given per adduct level. The error bars show the standard deviation. The matrix in SPE 1 and SPE 2 are based on hSA and sheep SA, respectively. There are no statistically significant differences between the experiments (p >0.05, calculated with one-way ANOVA)

Intensity, cps

Ratio of sample area (BPDE-His-Pro and BPDE– His-Pro-Tyr) and IS area (DBPDE-Lys)

E. Westberg et al.

0 Max. 2420.0 cps. 2000

BPDE-His-Pro 13 f mol/mg SRM m/z 555.3>257.0 Da

1000

0 Max. 340.0 cps. 300

BPDE-His-Pro-Tyr 19 f mol/mg

200

SRM m/z 718.3>257.0 Da

100 0

2

4

6

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

Time, min

Fig. 6 HPLC/MS-MS chromatograms from analysis of a calibration sample. Injection corresponding to 5 mg SA containing 65 fmol BPDE dipeptide and 95 fmol BPDE tripeptide, and with DBPDE-Lys (1,250 fmol) as IS. Sample prepared by enzymatic digestion and SPE enrichment of 10 mg SA with added standards of SA alkylated in vitro (BPDE-SA and DBPDE-SA in microgram amounts)

Analysis of bulky adducts to serum albumin with PAH diolepoxides

Serum albumin (SA) reaction with PAH-diolepoxide (PAHDE) (pH 7.5, 37 C) Using Eppendorf LoBind® tubes

Precipitation of the SA (pH 4, (NH4)2SO4 added, 20 min)

Wash of PAHDE-SA (MeOH, EtOAc and pentane)

PAHDE-SA 10 mg Addition of IS of another PAHDE-SA (50 mM NH4HCO3)

Enzymatic hydrolysis (20 mg SA/ml, 0.09 mg pronase/mg SA, 37ºC, over night)

Enrichment with SPE or HPLC (H2O:MeOH)

HPLC-MS/MS-analysis (ESI+, H2O:MeOH, 0.1% formic acid) Fig. 7 The analytical procedure established. The different steps in the work-up were evaluated, using a standard produced by in vitro alkylation of pure human SA. In the final procedure, 10 mg of SA was hydrolysed with pronase and as internal standard an amount of PAHDE-SA, produced from another PAH than the target PAH, was added. The enzymatic hydrolysate was enriched by HPLC or SPE before analysis by a triple quadrupole HPLC/MS-MS

BPDE. The observed adducts from DBPDE and DBADE showed the same type of analytes and the same MS fragmentation patterns as the BPDE adducts (Table 2) except that the -His-Pro-Tyr analyte was not detected in the hydrolysate of DBPDE-treated SA. This exception could possibly be explained by a different access for pronase to adduct-modified sites in the albumin. Evaluation of procedures used in the preparation and analysis of PAHDE adducts to SA With degradation using pronase, the adducted peptides BPDE-His-Pro and BPDE-His-Pro-Tyr were obtained in addition to BPDE-Lys. His, Pro and Tyr are somewhat bulky amino acids, and when located next to each other with a BPDE attached to His, the sequence probably constitutes a steric hindrance for cleavage by the enzyme. Efforts to obtain BPDE-His or BPDE-His-Pro as one analyte were undertaken

by the addition of RapiGest and papain to the enzymatic hydrolysate in different tests. However, no further improvement of the protein hydrolysis was achieved. Özbal et al. [20] mentioned en passant that enzymatic hydrolysis to His-Pro was complete at very low adduct levels (analysed by HPLC-LIF), which might be just below the LOD obtained in our study. A difference in hydrolysis efficiency might explain the observed variation in the relative peak areas of the respective analytes, both measured to vary between 40 and 60 % of the summed BPDE-His adducts. This observation supports the finding that the calibration lines showed higher reproducibility, and the r 2 values were higher when the area of the peaks of the di- and tripeptide analyte is summed. In our earlier work, hydrazine was used to obtain complete hydrolysis of BPDE-alkylated SA [17]. Hydrazine has some advantages, but given its toxicity, high reactivity and that it is dangerously unstable, it was concluded to be unsuitable for routine analysis. In the present study, alkaline hydrolysis of BPDE-SA was investigated to achieve complete degradation. Under the conditions evaluated, the desired BPDE-His as a single modified amino acid was obtained, with alkaline hydrolysis but a minor degradation of this analyte was also observed. These results, though, show that alkaline hydrolysis, seldom applied nowadays, could be of usage to obtain complete hydrolysis (optimal conditions in this study, 2 M NaOH, overnight, 100 °C), e.g. in work aiming at identification of stable adducts at high levels. In the present work, enzymatic hydrolysis using pronase was shown preferable as the LOD was considerably lower compared to alkaline hydrolysis, even though BPDE-His was obtained as the two different modified peptide analytes BPDE-His-Pro and BPDE-HisPro-Tyr after the digestion with pronase. Further evaluations concerned the use of Eppendorf Protein LoBind tubes which demonstrated a relatively large positive effect on the recovery. In the precipitation of SA (performed at low pH), the highest content of albumin in the precipitate was obtained after ∼20 min. After a prolonged time of precipitation, the albumin content in the precipitate decreased, probably due to increased amount of salt (the test conducted on pure SA). The level of BPDE-His-Pro was somewhat higher and of BPDE-His-Pro-Tyr somewhat lower after precipitation at pH 3 compared to neutral pH, but with regard to the sum of both analytes, no obvious loss of adducts was observed at the low pH. This indicated that the BPDE adducts were stable under the acidic conditions used during precipitation and adjustment to neutral pH was not required. Several, relatively simple procedures were investigated for the enrichment of the analytes from the hydrolysate. Among them, the butanol extraction, a method that has earlier been used for enrichment of PAH-DNA adducts [28], resulted in considerably lower recovery than enrichment by the HPLC or

E. Westberg et al.

SPE method. On the other hand, there was just a small difference in the recovery between HPLC and SPE. Based on solvent and time consumption associated to sample preparation, the SPE step is preferable to HPLC-UV, although the latter is most valuable in the initial detection of the analytes. Theoretically, the most suitable analyte(s) would be the PAH-modified amino acid(s) obtained after complete hydrolysis, because of less variation in the yield and a higher S/N ratio compared to a mixture of modified amino acids/peptides from uncomplete hydrolysis. The approach to use the sum of analytes and another PAHDE-modified SA as IS in the analysis was proven applicable and showed improved reproducibility in the analytical results. Such an IS could adjust for efficiency in enzymatic digestion, recovery in enrichment and for MS response. This also is a way to circumvent the need for synthesising stable isotope-substituted standards for each analyte, which would involve a comprehensive synthetic effort, particularly for standards of adducts from metabolites such as PAHDE. This especially concerns the stage of method development when it is still not decided which the most suitable analytes are. The quantification of adduct levels in the alkylated albumins used as standards was based on extinction coefficients for similar compounds of the specific PAHDE and HPLC-UV measurements. It was assumed that the approximate extinction coefficients used would not deviate more than a factor of two (see Material and Methods section) from the true values. By HPLC-UV and the equation established (Eq. 1), it was possible to quantify low amounts of adducts in a crude mixture from enzymatic digestion of alkylated SA standard. In the HPLC/MS-MS analysis matrix-based calibration curves were used which is essential in the analysis of biological samples. Since this work concerns PAH compounds, which are occurring ubiquitously in urban areas, the calibration samples were based on SA from sheep as the matrix, as it was assumed to have lower background level of PAHDE adducts than human SA. Parameters in the experimental procedures were tested to improve the LOD in the HPLC/MS-MS analysis. While increasing the amount of SA, the response in the MS analysis decreased. A reason may be a matrix effect or ion suppression which in this case was difficult to evaluate, as the standards are not available and probably will never be accessible in sufficient amounts for such tests. Lower amount of SA in the samples tested initially showed no large influence on the response (data not shown). The use of 10 mg SA resulted in the highest response of analytes. This is also the amount of SA which e.g. could be obtained from a mouse. Considering that very low adduct levels should be analysed, a lower amount of SA than 10 mg is not an option. Different HPLC/MS-MS systems were compared in order to evaluate the sensitivity. The type of the instrument was, as expected, an important factor for the LOD obtained.

Introduction of a column-switch setup enabled injections of larger volumes and pre-concentration of the analytes resulting in better sensitivity. In the newest HPLC/MS-MS system used, the instrumental detection was lower, and sample enrichment using a column-switch was not considered necessary. In future work, the use of a column-switch, enabling injection of the whole sample, should be taken into consideration. Furthermore, as applied in the present study, it is important to use several MRM transitions for each adduct in the MS/MS analyses in order to ensure correct analyte identification. The final LOD obtained with the developed method in combination with the HPLC/MS-MS setup was ca 1 fmol adduct/mg of the studied PAHDE-SA adducts, which means approximately seven adducts per 108 molecules of SA. Analysis of PAH adducts to human SA—comparison with other methods In a limited number of studies, adducts from BPDE bound as esters to SA (assumed to be (+)-anti-BPDE adducts) have been measured by MS methods as tetrols after their release by mild hydrolysis. Cohorts without occupational exposure (nonsmokers and smokers) showed mean adduct levels in the range 0.01–0.1 fmol/mg SA. A few studies could distinguish between controls and occupationally exposed persons (reviewed by Kaefferlein et al. [19]). For the present work, the most relevant comparison is a study by Özbal et al. [20], where stable His adducts were analysed with HPLC-LIF as BPDE-His-Pro analyte following enzymatic digestion of SA. The adduct levels ranged from

MS-MS analysis of bulky adducts to serum albumin with diolepoxides of polycyclic aromatic hydrocarbons as models.

Stable adducts to serum albumin (SA) from electrophilic and genotoxic compounds/metabolites can be used as biomarkers for quantification of the corres...
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