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A novel and reliable method for tetrahydrobiopterin quantification: Benzoyl chloride derivatization coupled with liquid chromatography-tandem mass spectrometry analysis
Author’s Accepted Manuscript A Novel and Reliable Method for Tetrahydrobiopterin Quantification: Benzoyl Chloride Derivatization Coupled with Liquid Chromatography-Tandem Mass Spectrometry Analysis Teng-Fei Yuan, Han-Qi Huang, Ling Gao, ShaoTing Wang, Yan Li
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To appear in: Free Radical Biology and Medicine Received date: 3 January 2018 Revised date: 2 February 2018 Accepted date: 26 February 2018 Cite this article as: Teng-Fei Yuan, Han-Qi Huang, Ling Gao, Shao-Ting Wang and Yan Li, A Novel and Reliable Method for Tetrahydrobiopterin Quantification: Benzoyl Chloride Derivatization Coupled with Liquid Chromatography-Tandem Mass Spectrometry Analysis, Free Radical Biology and Medicine, https://doi.org/10.1016/j.freeradbiomed.2018.02.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Novel and Reliable Method for Tetrahydrobiopterin Quantification: Benzoyl Chloride Derivatization Coupled with Liquid Chromatography-Tandem Mass Spectrometry Analysis Teng-Fei Yuan1, Han-Qi Huang1, Ling Gao, Shao-Ting Wang*, Yan Li* Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan 430060, China Tel.:+86-27-88041911; fax: +86-27-88041911. E-mail address: [email protected] ABSTRACT Tetrahydrobiopterin (BH4) is a crucial cofactor for nitric oxide synthase, acylglycerol mono-oxygenase and aromatic amino acids hydroxylases. Its significant function for redox pathways in vivo attracted much attention for long. However, because of the oxidizable and substoichiometric nature, analysis of BH4 has never been truly achieved with adequate sensitivity and applicability. In the present work, we pioneeringly stabilized BH4 by derivatizing the active secondary amine on five-position with benzoyl chloride (BC). Benefiting from the favorable chemical stability and excellent mass spectrometric sensitivity of the product (BH4-BC), ultra-sensitive and reliable quantification of endogenous BH4 in plasma was achieved using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In such methodology, BH4-BC-d5 was introduced as stable isotopic internal standard. And the limit of quantification (LOQ) could reach 0.02 ng mL-1. In the end, after investigation of plasma BH4 in healthy volunteers (n=38), we found that the levels of
1
These authors contributed equally to the present work. 1
BH4 were significantly and negatively correlated to age. Comparing with all the other existed strategies, the present method was obviously superior in sensitivity, specificity and practical applicability. It could be expected that this work could largely promote the future studies in BH4-related fields. Graphical abstract
Keywords: tetrahydrobiopterin; benzolyzation; mass spectrometry; human plasma
1. Introduction 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) was well known as the cofactor of phenylalanine-4-hydroxylase,
nitric
oxide
synthetase
and
glyceryl
ether
monooxygenase enzymes, which participated in many important redox processes in vivo (1). Profiting from the significant effects on adjusting phenylalanine metabolism, its synthetic form (6R-BH4, also named sapropterin) has been widely used to strengthen the phenylalanine tolerance for phenylketonuria (PKU) patients (2, 3). 2
Besides, BH4 played crucial roles in cardiovascular and mental diseases through regulating the production of the free radical nitric oxide and dopamine (4, 5). All these physiological functions were derived from its ultra-reducibility of the secondary amine on five-position (Figure 1). After oxidization, BH4 would be converted to the inactive forms, mainly dihydrobiopterin (BH2) and biopterin (B) (6). Obviously, evaluation of these biopterins (especially the active BH4) possessed significant clinical values for early diagnosis and individualized treatment of the related diseases. So far, various applicable methodologies have been carried out for BH2 and B quantification, including favorable MS-based methods (7, 8). However, analysis of BH4 was still challenging.
3
Fig. 1. Chemical structures of B, BH2, BH4, BH4-BC and BH4-BC-d5. And the MS/MS information of (I) BH4-BC and (II) BH4-BC-d5.
Presently, the most widely used method for BH4 quantification was still the indirect chemical oxidation (ICO) introduced by Fukushima and Nixon nearly forty years ago (9). It involved a pair of pH-differential iodine oxidation treatments and two corresponding HPLC-fluorimetric (FD) runs. Briefly, in acidic condition, BH4 and BH2 were both converted to B. And in basic condition, only BH2 was converted to B, while BH4 was oxidized to pterin. Hence, the different contents of B between two HPLC-FD runs could reflect the level of BH4. Apparently, such ICO method had many drawbacks in nature, which made it far from ideal for clinical usages. First, the acute oxidation processes would result in unknown byproducts so that the yields were unpredictable (10, 11). Second, the specificity of HPLC-FD was inadequate to eliminate the matrix effect. Meanwhile, this method was not compatible with the more specific MS detection platforms (12). Third, two independent HPLC-FD quantification processes were needed to calculate BH4 concentration, which would not only inevitably compromise the precision but also raise the experimental complexity and length. For improvements, online post-column conversion (OPC) strategies were also carried out and continuously modified since 1985 (13-16). Although they were direct methods, the uncommon online oxidizing reactors and electrochemical detectors impeded their popularization. More importantly, the mediocre specificity and 4
sensitivity made them far from ideal for complex bio-sample analysis. Benefitting from the favorable specificity and sensitivity, MS was considered as the most promising technique for pterin analysis (7, 8, 17). However, there were only four tentative works for BH4 quantification hitherto. The first attempt (by Foehr group) was still an ICO-based method, which involved an alkaline iodine-oxidation process (18). And the sensitivity (LOQ 5 ng mL-1) was not high enough for endogenous BH4 detection. Then Kim group carried out a direct MS method to analyze BH4 in rat brain (19). Nevertheless, no antioxidant was introduced in the study, which was inconsistent with all the other related researches (20, 21). Moreover, the use of non-isotopic internal standard (IS), epsilon-acetamidocaproic acid, was obviously undesirable for MS-quantification of BH4. The remaining two works by Fismen (22) and Arning (23) quantified BH4 in cell lysates and cerebrospinal fluid. They both utilized stable isotopic IS (15N-BH4) from Schircks Laboratories (Switzerland). Unfortunately, ascribing to the tedious synthesis process, such isotope was no longer commercially available. Besides, the introduction of ion-pairing reagent (heptafluorobutyric acid) in the mobile phase was not acceptable for most MS laboratories. From the above, ascribing to the inaccessibility of OPC and MS methods, most clinical researchers were still compromised to use the original ICO method for BH4 detection at present (24-26). So far, more than 650 citations have been made for such ICO method (data from Web of Science), while the reliability of the results has long been queried (27). Here, we described a robust and applicable benzoylation-based LC-MS/MS 5
method for BH4 quantification. Through simply derivatizing with benzoyl chloride (BC) (28, 29), BH4 was transformed to BH4-BC, which possessed much higher stability and MS sensitivity. Moreover, as the deuterated-BC (BC-d5) was commercially available, a synthetic isotopic IS (BH4-BC-d5) could be conveniently utilized. Profiting from these, comparing with all the existed methods, this work exhibited the best sensitivity (LOQ of 0.02 ng mL-1) and reliability. After analysis of plasma BH4 from healthy volunteers (n=38), a significant and negative correlation to the age was observed in the end.
2. Experimental section 2.1. CHEMICALS AND REAGENTS The standards of B (B2517-5mg), BH2 (37272-10mg), BH4 (T4425-5mg), BC (259950-100mL), BC-d5 (366048-1g), 1,4-dithioerythritol (DTE, D8255-5g), ethyl acetate (650528-1L) and hexane (34859-2.5L) were purchased from Sigma-Aldrich (Beijing, China). The ammonium carbonate (10001418-500g) was obtained from Sinopharm Chemical Reagent (Shanghai, China). Formic acid and acetonitrile (ACN) were of HPLC grade from Fischer Scientific (New Jersy, United States). The water used throughout the study was purified by a Milli-Q apparatus (Millipore, Bedford, MA). 2.2. PREPARATION OF STOCK SOLUTIONS, CALIBRATION AND QUALITY CONTROL SAMPLES DTE stock solution was prepared as 5% (w/w) in water for further dilution. Stock solutions of B, BH2 and BH4 (100 μg mL-1) were prepared in 50% ACN (v/v) in water 6
with 0.2% DTE (w/w). Further dilution for both calibration curve and quality control (QC) samples was performed using blank matrix with 0.2% DTE (w/w) in ice-water bath. The spiked concentrations of BH4 in calibration samples were 0.05, 0.1, 0.2, 0.5, 2.0, 5.0, 10.0 ng mL-1 with BH4-BC-d5 (1.0 ng mL-1). The spiked concentrations of BH4 in high-level, medium-level, low-level and LLOQ-level samples were 10.0, 1.0, 0.2 and 0.05 ng mL-1 respectively. All the stock solutions, calibration and QC samples were stored at -80 °C before use. The blank matrix was prepared by continuously exposing plasma to light for 8 hours.
The stock solution of IS (BH4-BC-d5) was synthesized using BH4 (100.0 ng
mL-1) and BC-d5 by the following described benzoylation process. Both blank matrix and IS solution were analyzed by the established benzoylation coupled LC-MS/MS protocol to exclude BH4 contamination. The whole study was supervised under the Ethics Committee of Renmin Hospital of Wuhan University. The consent procedure was based on the standard procedure. All the plasma samples were obtained from volunteers with permission. 2.3. SAMPLE PREPARATION The schematic of sample preparation was shown in Figure 2. Briefly, plasma was collected in EDTA-anticoagulant tubes from healthy volunteers. After collection, DTE was added immediately to 0.2% (w/w) for antioxidation. After centrifugation (3000g, 1 min at 4 °C), plasma (100 μL) was transferred into a polypropylene conical centrifuge tube in ice-water bath and IS solution (100.0 ng mL-1, 1 μL) was added. Then ice-water-cooled ACN (900 μL) was added following with vortexing (1 min) 7
and centrifugation (12,000 g, 2 min at 4 °C) for protein precipitation. The resulted supernatant (900 μL) was allowed to mix with ammonium carbonate (500 mM, 50 μL). Afterwards, BC (25 μL) was introduced and the solution was vortexed (5 min, at room temperature) for benzoylation. Then, ethyl acetate (500 μL) and hexane (300 μL) was added, vortexed (2 min) and centrifuged (3,000 g, 2 min) to eliminate benzoic acid. The subnatant was separated and dried (100 oC) under nitrogen gas. In the end, after reconstruction with ACN (50 μL) and centrifugation (12,000 g, 2 min), the supernatant was utilized for LC-MS/MS analysis. The inject volume was 20 μL.
Fig. 2. The schematic procedures of classic iodine chemical oxidation method and the present benzolyzation-based LC-MS/MS method for BH4 quantification.
2.4. LC-MS/MS ANALYSIS The LC–MS/MS platform consisted of an Ekspert ultraLC 100-XL system and an AB 8
SCIEX 4500 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA) equipped with an ESI source operating in the positive mode. The ESI inlet parameters were curtain gas (25.0), collision gas (medium), ionspray voltage (5500.0), temperature (500.0), ion source gas 1 (25.0) and ion source gas 2 (25.0). Data acquisition and processing were performed using AB SCIEX Analyst 1.6.2 Software (Applied Biosystems, Foster City, CA). As shown in Table S-1 (see online Supplementary material), the targets were monitored by multiple reaction monitoring (MRM) mode using the mass transitions (precursor ion → product ion) of BH4 (242.0 → 166.0), BH2 (240.0 → 165.0), B (238.1 → 220.0), BH4-BC (346.2 → 166.0 for quantification; 346.2 → 105.0 for qualitation) and BH4-BC-d5 (351.2 → 166.0 for quantification; 351.2 → 110.0 for qualitation). Additionally, the chemical structures of BH4-BC and BH4-BC-d5 were further confirmed with high resolution orbitrap mass spectrometry (LTQ Orbitrap MS, Thermo-Fisher Scientific, Waltham, MA, USA) as shown in Table S-2. The LC separation was performed under hydrophilic interaction chromatography (HILIC) mode on a Polar-Imidazole column (2.2 μm, 2.1 × 100 mm, Sepax Technologies, Newark, USA) with a flow rate of 0.35 mL min-1 at 40 °C. Fifteen percentages of ACN in water with 0.2% formic acid (v/v, Solution-A) and ACN with 0.2% formic acid (Solution-B) were employed as mobile phases. The gradient was 0– 3 min 98% B, 3–8 min 98–78% B, 8-8.1 min 78-98% B, 8.1-10 min 98% B. 2.5. METHOD DEVELOPMENT A series of parameters were optimized for sample preparation processes, including 9
solvents for protein precipitation, environment for benzolyzation, purification media for BH4-BC, etc. Besides, the benzolyzation mechanism between BH4 and BC was discussed. 2.6. METHOD VALIDATION 2.6.1. SELECTIVITY AND CARRY-OVER EFFECT The method selectivity was studied by investigating whether the blank plasma samples from different donors (n= 6) would cause interference toward targets. The carry-over effects were evaluated by analyzing the blank matrixes before and after injection of the upper limit of quantification samples (n=6). The residues should be less than 15% of the lower limit of quantification (LLOQ).
2.6.2. LINEARITY AND SENSITIVITY The calibration curves were calculated using the ratios of peak area of BH4-BC to BH4-BC-d5 versus the concentrations of BH4 spiked in plasma by a linear least squares regression model. The square of linear correlation coefficient (r2) should be higher than 0.99. The calibration was prepared at seven levels initially in duplicate: 0.05, 0.1, 0.2, 0.5, 2.0, 5.0, 10.0 ng mL-1for BH4. And the LOQ was calculated as the ratio of signal to noise of ten.
2.6.3. ACCURACY AND IMPRECISION Accuracy was calculated by dividing the measured BH4 concentrations to the nominal spiked values in plasma. And imprecision was the coefficient of variation (CV) of the 10
measurements. Both accuracy and imprecision were investigated on four spiked levels (high-, medium-, low- and LLOQ-level) with the concentrations of 10.0, 1.0, 0.2 and 0.05 ng mL-1 in the one day (intraday) and in six consecutive days (interday). The values of accuracy should be 85–115% (80-120% for LLOQ-level). And imprecision should not be higher than 15% (20% for LLOQ-level).
2.6.4. MATRIX EFFECT Matrix effect was evaluated by the CV of recoveries for six spiked blank matrixes from different donors. Samples were spiked for two levels (low- and high-level) to this end. The results should be less than 15%.
2.6.5. STABILITY Stability was carefully investigated on five aspects and evaluated by the recoveries (dividing the results from Group-X to Group-Control). (I) The stability of BH4 in standard solution. LLOQ-, low-, medium- and high-level (I-A, B, C and D) of BH4 standard solutions were prepared and analyzed immediately (I-Control) or stored at 25 °C (1, 4, 12 and 24 h), 4 °C (2, 12, 24 and 48 h) and -80 °C (7, 30 and 180 d) before analysis. Freeze-thaw stability was tested after three cycles of freezing (-80 °C) and thawing (25 °C). (II) The stability of BH4 during plasma separation. Blood samples (n=6) from different donors were studied. Each blood sample was divided into five parts (II-Control, A, B, C and D). II-Control was added 0.5% DTE (w/w) and then 11
centrifuged and analyzed. II-A to D were spiked with 0, 0.05%, 0.1% and 0.2% DTE (w/w) respectively and then centrifuged and analyzed. (III) The stability of BH4 in whole blood after collection. Blood samples (n=6) from different donors were studied. Each blood sample was divided into three parts (III-Control, A and B). III-Control was immediately spiked with 0.2% DTE (w/w) and analyzed. III-A and B were stored at 25 °C and 4 °C respectively, for 10, 30 and 60 min before further treatment. (IV) The stability of BH4 in plasma. Blank matrixes (n=6) from different donors with 0.2% DTE (w/w) were spiked with LLOQ-, low-, medium- and high-level (IV-A, B, C and D) of BH4 in ice-water bath. Samples were analyzed immediately (IV-Control) or stored at 25 °C (0.5, 1 and 4 h), 4 °C (1, 4 and 8 h) and -80 °C (7, 30 and 180 d) before analysis. Freeze-thaw stability was tested after one to three cycles of freezing (-80 °C) and thawing (25 °C). (V) The stability of BH4-BC and BH4-BC-d5. Blank matrixes (n=6) from different donors were spiked with LLOQ-, low-, medium- and high-level (V-A, B, C and D) of BH4 in ice-water bath and pretreated following the optimized protocol. Samples were analyzed immediately (V-Control) or stored at 25 °C (72 h), 4 °C (7 d) and -80 °C (180 d) respectively before analysis. Freeze-thaw stability was tested after three cycles of freezing (-80 °C) and thawing (25 °C).
3. Results and Discussion 3.1. METHOD DEVELOPMENT 12
The schematic of the proposed protocol was shown in Figure 2. As BC was sensitive to hydroxyl group, ACN was chosen for protein precipitation instead of alcohols. To neutralize the produced acid from BC hydrolyzation, several buffer solutions were tested including sodium carbonate, sodium tetraborate and ammonium carbonate. All of them showed acceptable performance. Ammonium carbonate was used because its product, ammonium chloride, was volatile so that more compatible to MS. Then, the amount of BC was investigated from 5 to 50 μL. We found that 25 μL of BC was adequate to complete the benzoylation process. To eliminate the interference from benzoic acid, different extracting solvents (ethyl acetate, hexane, butyl alcohol and methyl tert-butyl ether) were tested. Consequently, the combination of ethyl acetate and hexane (5/3, v/v) was chosen for not only its high efficiency for benzoic acid elimination but also the ability to induce phase separation between water and ACN. Benefiting from the hydrophilicity, BH4-BC was able to purify and concentrate in the subnatant water-phase. The mechanism of benzolyzation between BH4 and BC was investigated in the first place. As shown in Figure 1, there were four amino groups (position 2, 3, 5 and 8) in BH4, which could be potentially modified with BC. Comparingly, BH2 possessed three of them (position 2, 3 and 8) and B possessed two (position 2 and 3). After going through the pre-described reacting process, except for BH4-BC, neither BH2-BC nor B-BC could be identified by direct MS analysis. For further confirmation, we investigated the recoveries of BH2 and B after BC treatment (Supplementary material). To this end, another gradient for simultaneously monitoring BH4-BC as well as BH2 13
and B was used (0-3 min 95% B, 3-8 min 95-70% B, 8-12 min 70% B, 12-12.5 min 70-95% B, 12.5-15 min 95% B). A typical chromatogram was shown in Figure S-1. After BC treatment, no obvious consumption for BH2 and B was found (the recoveries were 92.1% for BH2 and 95.3% for B). Such result indicated benzolyzation could only take place on the 5-position amino group of BH4 in the proposed condition. The reason might be ascribed to the less active nature of amino groups on position 2, 3 and 8 as well as the block from the bulk ammonium carbonate.
3.2. METHOD VALIDATION 3.2.1. SELECTIVITY AND CARRY-OVER EFFECT Figure 3 showed the typical LC-MS/MS chromatograms of blank and spiked plasma samples. Obviously, there was no interference at the retention time of BH4 (7.68 min) in blank group (Figure 3-I), which indicated the excellent selectivity of the present method. Additionally, as no residual BH4–BC could be identified after analysis of upper limit of quantification samples (Figure 3-II), the carry-over effect was considered to be satisfactory.
14
Fig. 3. The benzolyzation-based LC-MS/MS analysis of (I) blank plasma sample; (II) blank plasma sample after injection of 10.0 ng mL-1 BH4-BC; (III) blank plasma sample spiked with LLOQ-level of BH4 (0.05 ng mL-1).
3.2.2 LINEARITY AND SENSITIVITY A linear range of 0.05–10.0 ng mL-1 was obtained with r2 higher than 0.99 (y=0.9210x-0.0244). The lowest concentration of the calibration curve was accepted as LLOQ, while the LOQ were calculated as 0.02 ng mL-1 (Table 1 and Figure S-2). Such sensitivity was at least one order of magnitudes higher than all the existed methods, which could be credited to the superior chemical stability and MS response of BH4-BC versus BH4. The typical LC-MS/MS chromatogram of LLOQ sample was shown as Figure 3-III. For comparison, plasma samples spiked with BH4 (50.0 ng 15
mL-1) were also detected directly by LC-MS/MS after protein precipitation (Figure S-3). As shown in Figure S-3-II, BH2, as one of the byproducts, was also monitored. In this case, it was obvious that majority of BH4 was degraded during LC separation (Peak 2) as well as ionization (Peak 1) processes. Ascribing to the degradation and also the lower MS sensitivity, the calculated LOQ for direct analysis (5.0 ng mL-1) was over 200 times higher than the present benzolyzation method.
Table 1. The retention time, linear regression and limit of quantification (LOQ) data for benzoylation-based LC-MS/MS method for BH4 quantification. Retention time Linear range
Calibration curves
LOQ
(min)
(ng mL-1)
Slope
Intercept
r2
(ng mL-1)
7.68
0.05-10.0
0.9210
-0.0244
0.9998
0.02
3.2.3. ACCURACY AND IMPRECISION As listed in Table S-3, the accuracy (expressed as recovery) was calculated as 83.0-86.0% for LLOQ-level and 86.0-95.0% for other levels. The imprecision (expressed as CV values) of LLOQ-level was 15.5% and 16.1% for intraday and interday, while the CV values of other levels were 7.4-12.1%. Such results demonstrated the excellent reliability of the present method on plasma BH4 determination.
3.2.4. MATRIX EFFECT 16
The average recoveries from different plasma matrixes were 91.8% and 95.1% for low- and high-level spiked groups. And the CV values were both lower than 10.1% (Table S-4), which indicated that with the correction from the isotopic IS (BH4-BC-d5), the interferences from different plasma matrixes would not affect the quantification of BH4. In addition, we also examined the data without normalization of isotopic IS (as absolute signal intensities). In this case, CV values were 37.8% and 41.5%, which demonstrated the unacceptable precision without isotopic IS correction.
3.2.5. STABILITY Because of the ultra-reducibility of BH4, the stability test during its quantification has long been a critical topic. Both of sample treatment and instrumental detection processes could cause degradation. As mentioned above, we carefully studied the stability from five parts. (I) The result of BH4 stability in standard solution was shown in Table S-5. It was indicated that BH4 could be well preserved with 0.2% DTE in standard solution for at least 12 h at 25 °C, 24 h at 4 °C and 180 d at -80 °C (recoveries higher than 85%). Additionally, after three cycles of freezing and thawing, little degradation was observed (recoveries 88.6-100.5%). (II) The stability of BH4 during plasma separation was significantly affected by DTE concentration. As shown in Table S-6, more than 90% of BH4 would be degraded without DTE protection during plasma separation. In contrast, after spiking 0.1% DTE or more before centrifugation, most BH4 could be preserved. Consequently, 17
0.2% DTE (w/w) was chosen as the optimum spiking concentration. (III) As there was no DTE-containing blood collector commercially available, the stability of BH4 in whole blood before DTE-spiking was studied. As shown in Table S-7, approximately 30% of BH4 lost in 10 min after storing at 25 °C, while over 80% of BH4 could be preserved for 30 min at 4 °C. These results suggested the necessity of low-temperature preservation for blood sample, if DTE could not be spiked immediately. (IV) The stability of BH4 in plasma was found obviously increased with the decrease of temperature (Table S-8). For instance, BH4 could be well preserved in plasma for 4 h at 4 °C with DTE (recovery around 85%), while nearly half of it would lose for only 1 h at 25 °C. When storing under −80 °C, no obvious degradation occurred for at least 6 months. As for freeze-thaw tests, three cycles would cause about 30% decrease of recovery, which indicated one should prevent repeated freeze-thaw operation during BH4 analysis. (V) The stability of BH4-BC and BH4-BC-d5 was highly increased comparing with BH4. As shown in Table S-9, in all the tested circumstances, the recoveries were ranging from 88.6-105.4%. Such excellent stability provided several advantages, including ensuring the reproducibility and applicability of the isotopic IS; making it possible to utilize diverse purification, separation and detection technologies; ensuring the accuracy and precision during analysis.
3.3. COMPARISON WITH OTHER METHODS 18
The typical strategies for BH4 analysis was concluded in Table S-10. As mentioned above, all the existed methods were suffered from some drawbacks in nature, which decreased their application values. Specifically, a comparison between the classic ICO method and the proposed benzolyzaion-based LC-MS/MS method was presented in Figure 2. Generally, four obvious advantages could be concluded for the present method. First, the present method provided reliable quantification through isotope dilution technique, which was more accurate and reproducible. Second, the MS detection was far more specific than FD detection. Third, the sensitivity was higher. Forth, the whole analyzing process was much simpler and faster.
4. Practical Application The present method was then applied to investigate the BH4 level in human plasma. In the study, we enrolled 38 healthy volunteers including 20 males and 18 females between 18 and 65 years old. The detail information for each volunteer was listed in Table S-11. One typical LC-MS/MS chromatogram was shown in Figure 4. In general, the concentration of plasma BH4 was ranging from 0.66 to 6.80 ng mL-1 (3.09 ± 1.43 ng mL-1). Such result was consistent with previous findings (30-32). After data analysis, as shown in Figure 5, we found that the levels of plasma BH4 were significantly and negatively correlated to the age via Pearson correlation analysis (r=-0.688, P<0.001) using SPSS version 20.0 (SPSS Inc, Chicago, IL). Such phenomenon may be associated to the change of redox status with age in vivo (33, 34). Accordingly, we suggested that the age may need to be treated as a significant 19
parameter in the further clinical-related studies based on plasma BH4. However, considering the limit of sample size, more systematic studies were still needed to elucidate such association.
Fig. 4. Typical LC-MS/MS chromatograms for plasma BH4 quantification for healthy volunteers.
20
Fig. 5. Results of Pearson correlation analysis, performed to evaluate the association between plasma concentration of BH4 and age.
5. Conclusion A highly sensitive and applicable LC-MS/MS method was established for quantification of BH4 in plasma. By using benzoyl chloride (BC) as derivatizing reagent, the ultra-active amino group on five position of BH4 was selectively stabilized for the very first time, which allowed BH4 (presented as BH4-BC) could be efficiently purified by diverse pretreatment techniques without degradation. Meanwhile, for replacement of the commercially unavailable isotopic BH4, BH4-BC-d5 was conveniently synthesized and performed perfectly in stable isotope dilution strategy for MS analysis. In the end, through quantification of plasma BH4 in healthy volunteers (n=38), we observed a significant and negative correlation between plasma BH4 levels and age. Comparing with all the other existed strategies, the 21
present method was obviously superior in sensitivity, specificity and practical applicability. We expected it could largely promote the future studies on BH4-related fields.
Acknowledgements The authors thank the Natural Science Foundation of China for financial support (21705121, 81572069, 81571376 and 81170767).
Supplementary material Available The authors declare that all the other data supporting the finding of this study are available within the article and its Supplementary material file and from the corresponding author on reasonable request.
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Highlights 1. BH4 is stabilized for the very first time by a simple benzoylization process. 2. Quantification of plasma BH4 is realized with ultra-sensitivity (LOQ 0.02 ng/mL). 3. Plasma BH4 is found negatively correlated with age from 38 healthy volunteers.
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S0891-5849(18)30092-3 https://doi.org/10.1016/j.freeradbiomed.2018.02.035 FRB13644
To appear in: Free Radical Biology and Medicine Received date: 3 January 2018 Revised date: 2 February 2018 Accepted date: 26 February 2018 Cite this article as: Teng-Fei Yuan, Han-Qi Huang, Ling Gao, Shao-Ting Wang and Yan Li, A Novel and Reliable Method for Tetrahydrobiopterin Quantification: Benzoyl Chloride Derivatization Coupled with Liquid Chromatography-Tandem Mass Spectrometry Analysis, Free Radical Biology and Medicine, https://doi.org/10.1016/j.freeradbiomed.2018.02.035 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Novel and Reliable Method for Tetrahydrobiopterin Quantification: Benzoyl Chloride Derivatization Coupled with Liquid Chromatography-Tandem Mass Spectrometry Analysis Teng-Fei Yuan1, Han-Qi Huang1, Ling Gao, Shao-Ting Wang*, Yan Li* Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan 430060, China Tel.:+86-27-88041911; fax: +86-27-88041911. E-mail address: [email protected] ABSTRACT Tetrahydrobiopterin (BH4) is a crucial cofactor for nitric oxide synthase, acylglycerol mono-oxygenase and aromatic amino acids hydroxylases. Its significant function for redox pathways in vivo attracted much attention for long. However, because of the oxidizable and substoichiometric nature, analysis of BH4 has never been truly achieved with adequate sensitivity and applicability. In the present work, we pioneeringly stabilized BH4 by derivatizing the active secondary amine on five-position with benzoyl chloride (BC). Benefiting from the favorable chemical stability and excellent mass spectrometric sensitivity of the product (BH4-BC), ultra-sensitive and reliable quantification of endogenous BH4 in plasma was achieved using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In such methodology, BH4-BC-d5 was introduced as stable isotopic internal standard. And the limit of quantification (LOQ) could reach 0.02 ng mL-1. In the end, after investigation of plasma BH4 in healthy volunteers (n=38), we found that the levels of
1
These authors contributed equally to the present work. 1
BH4 were significantly and negatively correlated to age. Comparing with all the other existed strategies, the present method was obviously superior in sensitivity, specificity and practical applicability. It could be expected that this work could largely promote the future studies in BH4-related fields. Graphical abstract
Keywords: tetrahydrobiopterin; benzolyzation; mass spectrometry; human plasma
1. Introduction 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) was well known as the cofactor of phenylalanine-4-hydroxylase,
nitric
oxide
synthetase
and
glyceryl
ether
monooxygenase enzymes, which participated in many important redox processes in vivo (1). Profiting from the significant effects on adjusting phenylalanine metabolism, its synthetic form (6R-BH4, also named sapropterin) has been widely used to strengthen the phenylalanine tolerance for phenylketonuria (PKU) patients (2, 3). 2
Besides, BH4 played crucial roles in cardiovascular and mental diseases through regulating the production of the free radical nitric oxide and dopamine (4, 5). All these physiological functions were derived from its ultra-reducibility of the secondary amine on five-position (Figure 1). After oxidization, BH4 would be converted to the inactive forms, mainly dihydrobiopterin (BH2) and biopterin (B) (6). Obviously, evaluation of these biopterins (especially the active BH4) possessed significant clinical values for early diagnosis and individualized treatment of the related diseases. So far, various applicable methodologies have been carried out for BH2 and B quantification, including favorable MS-based methods (7, 8). However, analysis of BH4 was still challenging.
3
Fig. 1. Chemical structures of B, BH2, BH4, BH4-BC and BH4-BC-d5. And the MS/MS information of (I) BH4-BC and (II) BH4-BC-d5.
Presently, the most widely used method for BH4 quantification was still the indirect chemical oxidation (ICO) introduced by Fukushima and Nixon nearly forty years ago (9). It involved a pair of pH-differential iodine oxidation treatments and two corresponding HPLC-fluorimetric (FD) runs. Briefly, in acidic condition, BH4 and BH2 were both converted to B. And in basic condition, only BH2 was converted to B, while BH4 was oxidized to pterin. Hence, the different contents of B between two HPLC-FD runs could reflect the level of BH4. Apparently, such ICO method had many drawbacks in nature, which made it far from ideal for clinical usages. First, the acute oxidation processes would result in unknown byproducts so that the yields were unpredictable (10, 11). Second, the specificity of HPLC-FD was inadequate to eliminate the matrix effect. Meanwhile, this method was not compatible with the more specific MS detection platforms (12). Third, two independent HPLC-FD quantification processes were needed to calculate BH4 concentration, which would not only inevitably compromise the precision but also raise the experimental complexity and length. For improvements, online post-column conversion (OPC) strategies were also carried out and continuously modified since 1985 (13-16). Although they were direct methods, the uncommon online oxidizing reactors and electrochemical detectors impeded their popularization. More importantly, the mediocre specificity and 4
sensitivity made them far from ideal for complex bio-sample analysis. Benefitting from the favorable specificity and sensitivity, MS was considered as the most promising technique for pterin analysis (7, 8, 17). However, there were only four tentative works for BH4 quantification hitherto. The first attempt (by Foehr group) was still an ICO-based method, which involved an alkaline iodine-oxidation process (18). And the sensitivity (LOQ 5 ng mL-1) was not high enough for endogenous BH4 detection. Then Kim group carried out a direct MS method to analyze BH4 in rat brain (19). Nevertheless, no antioxidant was introduced in the study, which was inconsistent with all the other related researches (20, 21). Moreover, the use of non-isotopic internal standard (IS), epsilon-acetamidocaproic acid, was obviously undesirable for MS-quantification of BH4. The remaining two works by Fismen (22) and Arning (23) quantified BH4 in cell lysates and cerebrospinal fluid. They both utilized stable isotopic IS (15N-BH4) from Schircks Laboratories (Switzerland). Unfortunately, ascribing to the tedious synthesis process, such isotope was no longer commercially available. Besides, the introduction of ion-pairing reagent (heptafluorobutyric acid) in the mobile phase was not acceptable for most MS laboratories. From the above, ascribing to the inaccessibility of OPC and MS methods, most clinical researchers were still compromised to use the original ICO method for BH4 detection at present (24-26). So far, more than 650 citations have been made for such ICO method (data from Web of Science), while the reliability of the results has long been queried (27). Here, we described a robust and applicable benzoylation-based LC-MS/MS 5
method for BH4 quantification. Through simply derivatizing with benzoyl chloride (BC) (28, 29), BH4 was transformed to BH4-BC, which possessed much higher stability and MS sensitivity. Moreover, as the deuterated-BC (BC-d5) was commercially available, a synthetic isotopic IS (BH4-BC-d5) could be conveniently utilized. Profiting from these, comparing with all the existed methods, this work exhibited the best sensitivity (LOQ of 0.02 ng mL-1) and reliability. After analysis of plasma BH4 from healthy volunteers (n=38), a significant and negative correlation to the age was observed in the end.
2. Experimental section 2.1. CHEMICALS AND REAGENTS The standards of B (B2517-5mg), BH2 (37272-10mg), BH4 (T4425-5mg), BC (259950-100mL), BC-d5 (366048-1g), 1,4-dithioerythritol (DTE, D8255-5g), ethyl acetate (650528-1L) and hexane (34859-2.5L) were purchased from Sigma-Aldrich (Beijing, China). The ammonium carbonate (10001418-500g) was obtained from Sinopharm Chemical Reagent (Shanghai, China). Formic acid and acetonitrile (ACN) were of HPLC grade from Fischer Scientific (New Jersy, United States). The water used throughout the study was purified by a Milli-Q apparatus (Millipore, Bedford, MA). 2.2. PREPARATION OF STOCK SOLUTIONS, CALIBRATION AND QUALITY CONTROL SAMPLES DTE stock solution was prepared as 5% (w/w) in water for further dilution. Stock solutions of B, BH2 and BH4 (100 μg mL-1) were prepared in 50% ACN (v/v) in water 6
with 0.2% DTE (w/w). Further dilution for both calibration curve and quality control (QC) samples was performed using blank matrix with 0.2% DTE (w/w) in ice-water bath. The spiked concentrations of BH4 in calibration samples were 0.05, 0.1, 0.2, 0.5, 2.0, 5.0, 10.0 ng mL-1 with BH4-BC-d5 (1.0 ng mL-1). The spiked concentrations of BH4 in high-level, medium-level, low-level and LLOQ-level samples were 10.0, 1.0, 0.2 and 0.05 ng mL-1 respectively. All the stock solutions, calibration and QC samples were stored at -80 °C before use. The blank matrix was prepared by continuously exposing plasma to light for 8 hours.
The stock solution of IS (BH4-BC-d5) was synthesized using BH4 (100.0 ng
mL-1) and BC-d5 by the following described benzoylation process. Both blank matrix and IS solution were analyzed by the established benzoylation coupled LC-MS/MS protocol to exclude BH4 contamination. The whole study was supervised under the Ethics Committee of Renmin Hospital of Wuhan University. The consent procedure was based on the standard procedure. All the plasma samples were obtained from volunteers with permission. 2.3. SAMPLE PREPARATION The schematic of sample preparation was shown in Figure 2. Briefly, plasma was collected in EDTA-anticoagulant tubes from healthy volunteers. After collection, DTE was added immediately to 0.2% (w/w) for antioxidation. After centrifugation (3000g, 1 min at 4 °C), plasma (100 μL) was transferred into a polypropylene conical centrifuge tube in ice-water bath and IS solution (100.0 ng mL-1, 1 μL) was added. Then ice-water-cooled ACN (900 μL) was added following with vortexing (1 min) 7
and centrifugation (12,000 g, 2 min at 4 °C) for protein precipitation. The resulted supernatant (900 μL) was allowed to mix with ammonium carbonate (500 mM, 50 μL). Afterwards, BC (25 μL) was introduced and the solution was vortexed (5 min, at room temperature) for benzoylation. Then, ethyl acetate (500 μL) and hexane (300 μL) was added, vortexed (2 min) and centrifuged (3,000 g, 2 min) to eliminate benzoic acid. The subnatant was separated and dried (100 oC) under nitrogen gas. In the end, after reconstruction with ACN (50 μL) and centrifugation (12,000 g, 2 min), the supernatant was utilized for LC-MS/MS analysis. The inject volume was 20 μL.
Fig. 2. The schematic procedures of classic iodine chemical oxidation method and the present benzolyzation-based LC-MS/MS method for BH4 quantification.
2.4. LC-MS/MS ANALYSIS The LC–MS/MS platform consisted of an Ekspert ultraLC 100-XL system and an AB 8
SCIEX 4500 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA) equipped with an ESI source operating in the positive mode. The ESI inlet parameters were curtain gas (25.0), collision gas (medium), ionspray voltage (5500.0), temperature (500.0), ion source gas 1 (25.0) and ion source gas 2 (25.0). Data acquisition and processing were performed using AB SCIEX Analyst 1.6.2 Software (Applied Biosystems, Foster City, CA). As shown in Table S-1 (see online Supplementary material), the targets were monitored by multiple reaction monitoring (MRM) mode using the mass transitions (precursor ion → product ion) of BH4 (242.0 → 166.0), BH2 (240.0 → 165.0), B (238.1 → 220.0), BH4-BC (346.2 → 166.0 for quantification; 346.2 → 105.0 for qualitation) and BH4-BC-d5 (351.2 → 166.0 for quantification; 351.2 → 110.0 for qualitation). Additionally, the chemical structures of BH4-BC and BH4-BC-d5 were further confirmed with high resolution orbitrap mass spectrometry (LTQ Orbitrap MS, Thermo-Fisher Scientific, Waltham, MA, USA) as shown in Table S-2. The LC separation was performed under hydrophilic interaction chromatography (HILIC) mode on a Polar-Imidazole column (2.2 μm, 2.1 × 100 mm, Sepax Technologies, Newark, USA) with a flow rate of 0.35 mL min-1 at 40 °C. Fifteen percentages of ACN in water with 0.2% formic acid (v/v, Solution-A) and ACN with 0.2% formic acid (Solution-B) were employed as mobile phases. The gradient was 0– 3 min 98% B, 3–8 min 98–78% B, 8-8.1 min 78-98% B, 8.1-10 min 98% B. 2.5. METHOD DEVELOPMENT A series of parameters were optimized for sample preparation processes, including 9
solvents for protein precipitation, environment for benzolyzation, purification media for BH4-BC, etc. Besides, the benzolyzation mechanism between BH4 and BC was discussed. 2.6. METHOD VALIDATION 2.6.1. SELECTIVITY AND CARRY-OVER EFFECT The method selectivity was studied by investigating whether the blank plasma samples from different donors (n= 6) would cause interference toward targets. The carry-over effects were evaluated by analyzing the blank matrixes before and after injection of the upper limit of quantification samples (n=6). The residues should be less than 15% of the lower limit of quantification (LLOQ).
2.6.2. LINEARITY AND SENSITIVITY The calibration curves were calculated using the ratios of peak area of BH4-BC to BH4-BC-d5 versus the concentrations of BH4 spiked in plasma by a linear least squares regression model. The square of linear correlation coefficient (r2) should be higher than 0.99. The calibration was prepared at seven levels initially in duplicate: 0.05, 0.1, 0.2, 0.5, 2.0, 5.0, 10.0 ng mL-1for BH4. And the LOQ was calculated as the ratio of signal to noise of ten.
2.6.3. ACCURACY AND IMPRECISION Accuracy was calculated by dividing the measured BH4 concentrations to the nominal spiked values in plasma. And imprecision was the coefficient of variation (CV) of the 10
measurements. Both accuracy and imprecision were investigated on four spiked levels (high-, medium-, low- and LLOQ-level) with the concentrations of 10.0, 1.0, 0.2 and 0.05 ng mL-1 in the one day (intraday) and in six consecutive days (interday). The values of accuracy should be 85–115% (80-120% for LLOQ-level). And imprecision should not be higher than 15% (20% for LLOQ-level).
2.6.4. MATRIX EFFECT Matrix effect was evaluated by the CV of recoveries for six spiked blank matrixes from different donors. Samples were spiked for two levels (low- and high-level) to this end. The results should be less than 15%.
2.6.5. STABILITY Stability was carefully investigated on five aspects and evaluated by the recoveries (dividing the results from Group-X to Group-Control). (I) The stability of BH4 in standard solution. LLOQ-, low-, medium- and high-level (I-A, B, C and D) of BH4 standard solutions were prepared and analyzed immediately (I-Control) or stored at 25 °C (1, 4, 12 and 24 h), 4 °C (2, 12, 24 and 48 h) and -80 °C (7, 30 and 180 d) before analysis. Freeze-thaw stability was tested after three cycles of freezing (-80 °C) and thawing (25 °C). (II) The stability of BH4 during plasma separation. Blood samples (n=6) from different donors were studied. Each blood sample was divided into five parts (II-Control, A, B, C and D). II-Control was added 0.5% DTE (w/w) and then 11
centrifuged and analyzed. II-A to D were spiked with 0, 0.05%, 0.1% and 0.2% DTE (w/w) respectively and then centrifuged and analyzed. (III) The stability of BH4 in whole blood after collection. Blood samples (n=6) from different donors were studied. Each blood sample was divided into three parts (III-Control, A and B). III-Control was immediately spiked with 0.2% DTE (w/w) and analyzed. III-A and B were stored at 25 °C and 4 °C respectively, for 10, 30 and 60 min before further treatment. (IV) The stability of BH4 in plasma. Blank matrixes (n=6) from different donors with 0.2% DTE (w/w) were spiked with LLOQ-, low-, medium- and high-level (IV-A, B, C and D) of BH4 in ice-water bath. Samples were analyzed immediately (IV-Control) or stored at 25 °C (0.5, 1 and 4 h), 4 °C (1, 4 and 8 h) and -80 °C (7, 30 and 180 d) before analysis. Freeze-thaw stability was tested after one to three cycles of freezing (-80 °C) and thawing (25 °C). (V) The stability of BH4-BC and BH4-BC-d5. Blank matrixes (n=6) from different donors were spiked with LLOQ-, low-, medium- and high-level (V-A, B, C and D) of BH4 in ice-water bath and pretreated following the optimized protocol. Samples were analyzed immediately (V-Control) or stored at 25 °C (72 h), 4 °C (7 d) and -80 °C (180 d) respectively before analysis. Freeze-thaw stability was tested after three cycles of freezing (-80 °C) and thawing (25 °C).
3. Results and Discussion 3.1. METHOD DEVELOPMENT 12
The schematic of the proposed protocol was shown in Figure 2. As BC was sensitive to hydroxyl group, ACN was chosen for protein precipitation instead of alcohols. To neutralize the produced acid from BC hydrolyzation, several buffer solutions were tested including sodium carbonate, sodium tetraborate and ammonium carbonate. All of them showed acceptable performance. Ammonium carbonate was used because its product, ammonium chloride, was volatile so that more compatible to MS. Then, the amount of BC was investigated from 5 to 50 μL. We found that 25 μL of BC was adequate to complete the benzoylation process. To eliminate the interference from benzoic acid, different extracting solvents (ethyl acetate, hexane, butyl alcohol and methyl tert-butyl ether) were tested. Consequently, the combination of ethyl acetate and hexane (5/3, v/v) was chosen for not only its high efficiency for benzoic acid elimination but also the ability to induce phase separation between water and ACN. Benefiting from the hydrophilicity, BH4-BC was able to purify and concentrate in the subnatant water-phase. The mechanism of benzolyzation between BH4 and BC was investigated in the first place. As shown in Figure 1, there were four amino groups (position 2, 3, 5 and 8) in BH4, which could be potentially modified with BC. Comparingly, BH2 possessed three of them (position 2, 3 and 8) and B possessed two (position 2 and 3). After going through the pre-described reacting process, except for BH4-BC, neither BH2-BC nor B-BC could be identified by direct MS analysis. For further confirmation, we investigated the recoveries of BH2 and B after BC treatment (Supplementary material). To this end, another gradient for simultaneously monitoring BH4-BC as well as BH2 13
and B was used (0-3 min 95% B, 3-8 min 95-70% B, 8-12 min 70% B, 12-12.5 min 70-95% B, 12.5-15 min 95% B). A typical chromatogram was shown in Figure S-1. After BC treatment, no obvious consumption for BH2 and B was found (the recoveries were 92.1% for BH2 and 95.3% for B). Such result indicated benzolyzation could only take place on the 5-position amino group of BH4 in the proposed condition. The reason might be ascribed to the less active nature of amino groups on position 2, 3 and 8 as well as the block from the bulk ammonium carbonate.
3.2. METHOD VALIDATION 3.2.1. SELECTIVITY AND CARRY-OVER EFFECT Figure 3 showed the typical LC-MS/MS chromatograms of blank and spiked plasma samples. Obviously, there was no interference at the retention time of BH4 (7.68 min) in blank group (Figure 3-I), which indicated the excellent selectivity of the present method. Additionally, as no residual BH4–BC could be identified after analysis of upper limit of quantification samples (Figure 3-II), the carry-over effect was considered to be satisfactory.
14
Fig. 3. The benzolyzation-based LC-MS/MS analysis of (I) blank plasma sample; (II) blank plasma sample after injection of 10.0 ng mL-1 BH4-BC; (III) blank plasma sample spiked with LLOQ-level of BH4 (0.05 ng mL-1).
3.2.2 LINEARITY AND SENSITIVITY A linear range of 0.05–10.0 ng mL-1 was obtained with r2 higher than 0.99 (y=0.9210x-0.0244). The lowest concentration of the calibration curve was accepted as LLOQ, while the LOQ were calculated as 0.02 ng mL-1 (Table 1 and Figure S-2). Such sensitivity was at least one order of magnitudes higher than all the existed methods, which could be credited to the superior chemical stability and MS response of BH4-BC versus BH4. The typical LC-MS/MS chromatogram of LLOQ sample was shown as Figure 3-III. For comparison, plasma samples spiked with BH4 (50.0 ng 15
mL-1) were also detected directly by LC-MS/MS after protein precipitation (Figure S-3). As shown in Figure S-3-II, BH2, as one of the byproducts, was also monitored. In this case, it was obvious that majority of BH4 was degraded during LC separation (Peak 2) as well as ionization (Peak 1) processes. Ascribing to the degradation and also the lower MS sensitivity, the calculated LOQ for direct analysis (5.0 ng mL-1) was over 200 times higher than the present benzolyzation method.
Table 1. The retention time, linear regression and limit of quantification (LOQ) data for benzoylation-based LC-MS/MS method for BH4 quantification. Retention time Linear range
Calibration curves
LOQ
(min)
(ng mL-1)
Slope
Intercept
r2
(ng mL-1)
7.68
0.05-10.0
0.9210
-0.0244
0.9998
0.02
3.2.3. ACCURACY AND IMPRECISION As listed in Table S-3, the accuracy (expressed as recovery) was calculated as 83.0-86.0% for LLOQ-level and 86.0-95.0% for other levels. The imprecision (expressed as CV values) of LLOQ-level was 15.5% and 16.1% for intraday and interday, while the CV values of other levels were 7.4-12.1%. Such results demonstrated the excellent reliability of the present method on plasma BH4 determination.
3.2.4. MATRIX EFFECT 16
The average recoveries from different plasma matrixes were 91.8% and 95.1% for low- and high-level spiked groups. And the CV values were both lower than 10.1% (Table S-4), which indicated that with the correction from the isotopic IS (BH4-BC-d5), the interferences from different plasma matrixes would not affect the quantification of BH4. In addition, we also examined the data without normalization of isotopic IS (as absolute signal intensities). In this case, CV values were 37.8% and 41.5%, which demonstrated the unacceptable precision without isotopic IS correction.
3.2.5. STABILITY Because of the ultra-reducibility of BH4, the stability test during its quantification has long been a critical topic. Both of sample treatment and instrumental detection processes could cause degradation. As mentioned above, we carefully studied the stability from five parts. (I) The result of BH4 stability in standard solution was shown in Table S-5. It was indicated that BH4 could be well preserved with 0.2% DTE in standard solution for at least 12 h at 25 °C, 24 h at 4 °C and 180 d at -80 °C (recoveries higher than 85%). Additionally, after three cycles of freezing and thawing, little degradation was observed (recoveries 88.6-100.5%). (II) The stability of BH4 during plasma separation was significantly affected by DTE concentration. As shown in Table S-6, more than 90% of BH4 would be degraded without DTE protection during plasma separation. In contrast, after spiking 0.1% DTE or more before centrifugation, most BH4 could be preserved. Consequently, 17
0.2% DTE (w/w) was chosen as the optimum spiking concentration. (III) As there was no DTE-containing blood collector commercially available, the stability of BH4 in whole blood before DTE-spiking was studied. As shown in Table S-7, approximately 30% of BH4 lost in 10 min after storing at 25 °C, while over 80% of BH4 could be preserved for 30 min at 4 °C. These results suggested the necessity of low-temperature preservation for blood sample, if DTE could not be spiked immediately. (IV) The stability of BH4 in plasma was found obviously increased with the decrease of temperature (Table S-8). For instance, BH4 could be well preserved in plasma for 4 h at 4 °C with DTE (recovery around 85%), while nearly half of it would lose for only 1 h at 25 °C. When storing under −80 °C, no obvious degradation occurred for at least 6 months. As for freeze-thaw tests, three cycles would cause about 30% decrease of recovery, which indicated one should prevent repeated freeze-thaw operation during BH4 analysis. (V) The stability of BH4-BC and BH4-BC-d5 was highly increased comparing with BH4. As shown in Table S-9, in all the tested circumstances, the recoveries were ranging from 88.6-105.4%. Such excellent stability provided several advantages, including ensuring the reproducibility and applicability of the isotopic IS; making it possible to utilize diverse purification, separation and detection technologies; ensuring the accuracy and precision during analysis.
3.3. COMPARISON WITH OTHER METHODS 18
The typical strategies for BH4 analysis was concluded in Table S-10. As mentioned above, all the existed methods were suffered from some drawbacks in nature, which decreased their application values. Specifically, a comparison between the classic ICO method and the proposed benzolyzaion-based LC-MS/MS method was presented in Figure 2. Generally, four obvious advantages could be concluded for the present method. First, the present method provided reliable quantification through isotope dilution technique, which was more accurate and reproducible. Second, the MS detection was far more specific than FD detection. Third, the sensitivity was higher. Forth, the whole analyzing process was much simpler and faster.
4. Practical Application The present method was then applied to investigate the BH4 level in human plasma. In the study, we enrolled 38 healthy volunteers including 20 males and 18 females between 18 and 65 years old. The detail information for each volunteer was listed in Table S-11. One typical LC-MS/MS chromatogram was shown in Figure 4. In general, the concentration of plasma BH4 was ranging from 0.66 to 6.80 ng mL-1 (3.09 ± 1.43 ng mL-1). Such result was consistent with previous findings (30-32). After data analysis, as shown in Figure 5, we found that the levels of plasma BH4 were significantly and negatively correlated to the age via Pearson correlation analysis (r=-0.688, P<0.001) using SPSS version 20.0 (SPSS Inc, Chicago, IL). Such phenomenon may be associated to the change of redox status with age in vivo (33, 34). Accordingly, we suggested that the age may need to be treated as a significant 19
parameter in the further clinical-related studies based on plasma BH4. However, considering the limit of sample size, more systematic studies were still needed to elucidate such association.
Fig. 4. Typical LC-MS/MS chromatograms for plasma BH4 quantification for healthy volunteers.
20
Fig. 5. Results of Pearson correlation analysis, performed to evaluate the association between plasma concentration of BH4 and age.
5. Conclusion A highly sensitive and applicable LC-MS/MS method was established for quantification of BH4 in plasma. By using benzoyl chloride (BC) as derivatizing reagent, the ultra-active amino group on five position of BH4 was selectively stabilized for the very first time, which allowed BH4 (presented as BH4-BC) could be efficiently purified by diverse pretreatment techniques without degradation. Meanwhile, for replacement of the commercially unavailable isotopic BH4, BH4-BC-d5 was conveniently synthesized and performed perfectly in stable isotope dilution strategy for MS analysis. In the end, through quantification of plasma BH4 in healthy volunteers (n=38), we observed a significant and negative correlation between plasma BH4 levels and age. Comparing with all the other existed strategies, the 21
present method was obviously superior in sensitivity, specificity and practical applicability. We expected it could largely promote the future studies on BH4-related fields.
Acknowledgements The authors thank the Natural Science Foundation of China for financial support (21705121, 81572069, 81571376 and 81170767).
Supplementary material Available The authors declare that all the other data supporting the finding of this study are available within the article and its Supplementary material file and from the corresponding author on reasonable request.
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Highlights 1. BH4 is stabilized for the very first time by a simple benzoylization process. 2. Quantification of plasma BH4 is realized with ultra-sensitivity (LOQ 0.02 ng/mL). 3. Plasma BH4 is found negatively correlated with age from 38 healthy volunteers.
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