An Innovative Approach to the Preparation of Plasma Samples for

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An Innovative Approach to the Preparation of Plasma Samples for UHPLC−MS Analysis Michael Kaiser, Bartosch Lacheta, Maike Passon,* and Andreas Schieber

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Department of Nutritional and Food Sciences, Molecular Food Technology, University of Bonn, Endenicher Allee 19b, 53115 Bonn, Germany ABSTRACT: A new sample processing method for analyzing flavonol metabolites in plasma using enzymatic proteolysis was developed and validated. Four endopeptidases were examined regarding their influence on the analyte recovery of quercetin-3O-glucuronide (Q3GlcA). Methanol was added to inactivate and precipitate the enzymes, and samples were concentrated via evaporation prior to UHPLC−MS analysis. Quercetin-3-O-rutinoside (Q3Rut) was used as an internal standard. The selectivity and accuracy of the established UHPLC−ESI−MSn method showed a coefficient of variation (CV) of the repeatability of the measuring instrument of 1.7% for Q3GlcA. The average recovery of Q3GlcA was approximately 67% with an interday method precision of 24% and r = 46.9 as its repeatability. Therefore, enzymatic proteolysis has proven to be a suitable alternative to the methods previously described in the literature, such as solid-phase extraction (SPE). Still, the method has only been validated for Q3GlcA, but its applicability to other substance classes seems possible. KEYWORDS: flavonol glycosides, plasma samples, metabolites, proteolysis, UHPLC−MS analysis

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not found in plasma,13 it turned out later that the glycosides can be found in both plasma and urine.14 The aglycone quercetin could not be detected in plasma.15 Quercetin-3-Oglucuronide (Q3GlcA), quercetin-3′-O-sulfate, and isorhamnetin-3-O-glucuronide have been found to be the predominant metabolites of hepatic phase II conversions.16 These metabolites enter the organism via the bloodstream. Therefore, the analysis of blood plasma is an integral part of the elucidation of polyphenol metabolism. However, plasma is a highly complex matrix and a challenge with respect to sample preparation. Except for water, proteins represent the largest fraction, their content varying between individuals but usually amounting to 65−80 g/L.17,18 The plasma proteome is composed of approximately 300 proteins, with human serum albumin (HSA) being the major constituent at quantities of 40−50 g/L,17 followed by the globulins.18 In addition, bioactive proteins such as enzymes and peptide hormones are present, which may be influenced by interaction with phenolic compounds; for example, serine proteases may be inhibited by flavonols.19 Flavonoid metabolites have been shown to be moderate ligands of proteins. They bind preferentially to the binding site II of HSA. For quercetin derivatives, it was shown that several amino acids participate in the bonding process and the quercetin−HSA complex formed was stabilized by an Hbonding network at site I in subdomain IIA, causing a change in the protein secondary structure.20 This binding is known to build up very fast, as in the presence of quercetin derivatives, free HSA exists only for about 5−10 ns.21 It is assumed that in plasma approximately 90% of flavonoid metabolites are bound

INTRODUCTION Plant phenols including flavonoids occur in most higher plants and are thus part of our daily diet. Because of their putative benefits for human health, the scientific interest in these secondary plant metabolites is still ongoing. Total flavonoid intake is largely dependent on the particular diet of people. In this context, various studies carried out worldwide have tried to assess the total daily intake of flavonoids via food frequency questionnaires using the USDA database or other food databases but provide only estimated amounts.1,2 Because of its widespread occurrence, quercetin and its glycosides are ubiquitous in human nutrition, accounting for approximately 75% of the worldwide daily flavonoid intake.3 Several positive effects of flavonoids on human health have been reported recently, but so far, only antihypertensive, antiallergenic, anticancer, and cardioprotective properties were confirmed in vivo.4−8 In addition, it was demonstrated that flavonoids such as quercetin may not only cross the blood− brain barrier9 but may even affect the brain and central nervous system.10 These findings entailed the creation of databases to associate the observed health effects with individual phytochemicals, for example, flavonoids such as quercetin and its derivatives.11 The transfer of results obtained in animal or in vitro studies to humans is critical because both the bioavailability and the metabolism of flavonoids may affect their efficacy and may also differ from animal models because of differing enzymes and mechanisms of absorption. Therefore, studies related to the metabolic pathways, along with the identification and quantification of the metabolites, are of utmost importance. Quercetin is present in plants mainly in its glycosidic form and bound to glucose but also to rutinose. During their passage through the intestinal tract, the quercetin glycosides are hydrolyzed primarily by enzymes, causing a loss of the sugar moieties.12 Whereas in older studies quercetin glycosides were © 2019 American Chemical Society

Received: Revised: Accepted: Published: 6665

March 21, 2019 May 17, 2019 May 21, 2019 May 22, 2019 DOI: 10.1021/acs.jafc.9b01782 J. Agric. Food Chem. 2019, 67, 6665−6671

Article

Journal of Agricultural and Food Chemistry to HSA21,22 due to its high binding efficiency, which increases with decreasing temperature below 20 °C.22 The experiments of Boulton and co-workers lead to the assumption that at 37 °C more than 97% and at 20 °C more than 99% of quercetin was bound to HSA.22 As a result, it is important that the flavonoid metabolites be released from the proteins during sample preparation prior to LC−MS analysis. Otherwise, the results would lead to misinterpretations of the bioavailability, as the determined plasma concentration was poor. In metabolism studies, both liquid−liquid extraction (LLE) after precipitation of proteins with organic solvents and solid-phase extraction (SPE) have so far been used to extract the metabolites from untreated plasma.23,24 While both methods are capable of removing the proteins from the complex matrix, bound metabolites might only insufficiently be released, as neither LLE nor SPE are used to break the bond between HSA and the flavonols. In view of the predominantly proteinaceous nature of the plasma matrix, it is surprising that the enzymatic degradation of the proteins has so far not been taken into consideration. Therefore, the objective of this study was to develop a novel method for sample preparation based on enzymatic proteolysis. It was hypothesized that the decomposition of the primary structure of the proteins will lead to the release of noncovalently bound metabolites. By preceding enzymatic proteolysis, protein-bound metabolites should subsequently be extractable via the conducted LLE and therefore analyzable via LC−MS. For this purpose, four proteases, that is, pepsin, papain, trypsin, and protease type XIV from Streptomyces griseus (PXIV), were used during method development regarding the best possible analyte recovery. The crucial criterion to choose these enzymes was that they are already well-known and characterized regarding their structure, and main and side activities. After optimizing the enzyme-toprotein ratios to improve recovery, the method was employed for the determination of Q3GlcA using UHPLC−MS and validated.

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ammonium hydrogen carbonate buffer solution (pH 8.2) was used to adjust the pH of the samples.26 Papain (4 g/L) was dissolved in a 5 mM aqueous solution of L-cysteine (pH 6.5), and PXIV (4 g/L) was dissolved in purified water (pH 7). Quercetin (50 mg/L) was dissolved in aqueous methanol (50%, v/v). The stock solutions of Q3GlcA (50 mg/L) and Q3Rut (50 mg/L) were prepared in 20% (v/ v) methanol in water. Sample Preparation. To simulate in vivo conditions as realistically as possible, plasma samples were spiked with analyte concentrations of 1 or 5 μg/mL. For this purpose, 20 mL of plasma was spiked with the analyte, homogenized carefully to prevent protein denaturation by shear, and then allowed to rest for 30 min at room temperature to enable interactions between the analyte and plasma components as they would occur in vivo.21,22 The binding of quercetin derivatives to HSA occurs both at room temperature and at body temperature, as it is a spontaneous endothermic association22 taking place in a few nanoseconds.21 The resting time of 30 min was chosen on the basis of practical aspects after it had been shown in preliminary experiments that various time intervals tested (10, 20, 30, 45, and 60 min) had no significant influence on the recovery, suggesting the formation of a stable equilibrium22 between HSAbound and non-HSA-bound quercetin derivatives. To 1 mL of plasma containing the analyte and 3 μg of Q3Rut as an internal standard, 42 μL of formic acid was added to adjust the pH to approximately 2. Subsequently, 40 μL of pepsin (400 mg/L) was added, resulting in a calculated enzyme-to-protein ratio of 1:5000 (w/ w). Q3Rut was used as an internal standard because it fulfilled the requirements concerning the chemical similarity to the analyte, and because it is commercially available. After incubation of the samples for 20 h at 37 °C, the 2.5-fold amount of methanol was added to inactivate the enzymes and to precipitate remaining peptides. The samples then were homogenized and centrifuged at 17 000g. The supernatant was evaporated at 30 °C under a constant nitrogen stream to approximately one-half of the initial plasma volume. The samples were filtered through Chromafil RC 20/15 MS membrane filters with a pore width of 0.20 μm (Macherey-Nagel, Düren, Germany) before LC−MS analysis. During method development, plasma or aqueous samples were spiked with the analyte at concentrations of 1 and 5 μg/mL. The analysis was executed in duplicate or in triplicate, and the results were averaged. Plasma and aqueous samples were spiked with either quercetin or Q3GlcA. Aqueous samples were used to assess the influence of the enzyme on the analyte. The solutions and buffers required for dissolution of the different enzymes, but not the enzymes themselves, were added to spiked aqueous samples to evaluate their influence on the analyte. Furthermore, different enzyme-to-protein ratios were tested, ranging from 1:50 to 1:10 000 (w/w). These ratios were used because an enzyme-to-protein (w/w) ratio of 1:50 was stated in the distributor’s instructions as the standard procedure for pepsin to guarantee sufficient and quick proteolysis. For the calculation of the enzyme amounts required, a plasma protein concentration of about 80 g/L was assumed.17,18 Enzymes were inactivated either thermally at 80 °C for 5 min, or by addition of different organic solvents combined with the precipitation of the remaining peptides. The supernatant was evaporated either to dryness and dissolved in 1 mL of the solvent of the stock solution, or to remaining volumes of approximately 1000 or 500 μL. A control run was performed to ensure the stability of Q3GlcA in water at pH 7 during 20 h of incubation at 37 °C as well as during thermal enzyme inactivation at 80 °C. Another control showed that the internal standard Q3Rut was not affected either by these conditions. For the first series of experiments with plasma, a nonmatrixmatched aqueous calibration was used because too many parameters would have to be adjusted in each case, which in turn would have necessitated several calibrations with high consumption of plasma and reference substances. Therefore, a matrix-matched calibration was used only after the method development was finalized and the validation was carried out. The plasma for the matrix-matched calibration was previously prepared analogously to the samples. In brief, the nonspiked plasma was hydrolyzed by the use of pepsin,

MATERIALS AND METHODS

Chemicals and Reagents. Pepsin from porcine mucosa (63 units/mg), trypsin from porcine pancreas (>13.000 BAEE (Nαbenzoyl-L-arginine ethyl ester) units/mg), protease type XIV from Streptomyces griseus (5.2 units/mg), formic acid, urea, ammonium hydrogen carbonate, dimethyl sulfoxide, L-cysteine, and quercetin (>95%) were purchased from Sigma-Aldrich (Steinheim, Germany). Papain (3.1 units/mg) was supplied by Fluka (Buchs, Switzerland). The aqueous ammonia solution (25%) was obtained from Carl Roth (Karlsruhe, Germany). Water was purified using a Purelab Flex Water Purification System from ELGA LabWater (High Wycombe, UK). Acetonitrile, methanol, and ethanol were HPLC-grade and provided by Th. Geyer (Renningen, Germany). HPLC-grade 2-propanol was purchased from VWR Chemicals (Fontenay-sous-Bois, France). Dimethylformamide (p.a.) was supplied by Fisher Chemical (Loughborough, UK). Quercetin-3-O-rutinoside (rutin) (>95%) was obtained from Carl Roth (Karlsruhe, Germany), and quercetin-3-Oglucuronide was isolated25 from an extract of Riesling leaves (Vitis vinifera subsp. vinifera cv. Riesling) with a chromatographic purity of 97.5% (CV = 0.01%) at λ = 280 nm. All solvents were LC−MS grade. Acetonitrile and water were supplied by Th. Geyer (Renningen, Germany), and formic acid was purchased from Sigma-Aldrich (Steinheim, Germany). Human plasma was provided by the University Hospital Bonn, Germany. Preparation of Stock Solutions. The stock solution of pepsin (400 mg/L) was prepared in aqueous formic acid (1%, v/v, pH 2). Trypsin (4 g/L) was dissolved in 2 M aqueous urea. A 250 mM 6666

DOI: 10.1021/acs.jafc.9b01782 J. Agric. Food Chem. 2019, 67, 6665−6671

Journal of Agricultural and Food Chemistry

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precipitated using methanol, and the supernatant was evaporated to about one-half of the initial plasma volume. UHPLC−MS Analysis. For UHPLC−MSn analysis, an Acquity IClass UPLC system from Waters (Milford, MA) consisting of an Acquity I-Class UPLC Binary Solvent Manager, an Acquity UPLC Sample Manager-FL, and an Acquity UPLC PDA eλ detector was used. The UHPLC sample manager was cooled at 10 °C, and the column oven was set at 40 °C. For chromatographic separation, a Kinetex Phenylhexyl column (2.1 × 50 mm, 1.7 μm) from Phenomenex (Aschaffenburg, Germany) equipped with a VanGuard Acquity CSH Phenylhexyl precolumn (5 × 2.1 mm, 1.7 μm) from Waters (Milford, MA) was used. Eluent A was 0.1% formic acid in water; 0.1% formic acid in acetonitrile was used as eluent B. The flow rate was 0.6 mL/min for the following linear gradient: 0 min 6% B, 1.5 min 13% B, 3 min 20.5% B, 4 min 35.6% B, 4.3 min 100% B, 5 min 100% B, 5.3 min 6% B, and 6.3 min 6% B. The injection volume was 5 μL. The MS system was an LTQ XL Ion Trap MSn from Thermo Scientific (Waltham, MA). The spray voltage was 3.5 kV, the capillary temperature was set at 350 °C, and the capillary voltage was −31 V with the tube lens voltage at −95 V. An electrospray interface was used operating in the negative ionization mode. The instrument was tuned automatically using Q3GlcA. The MS was used in SRM mode with collision-induced dissociation (CID) for both Q3GlcA and Q3Rut. In both cases, the losses of the sugar moieties were induced using a normalized collision energy of 20% for Q3GlcA, [m/z] 477 → 301; and of 25% for Q3Rut, [m/z] 609 → 301. As software Xcalibur Version 2.2.0.48 from Thermo Fisher Scientific (Waltham, MA) was used. Validation. Linearity, Limits of Detection and Quantification. Calibration curves were established using plasma that had been enzymatically hydrolyzed and precipitated using methanol, and that was spiked with Q3GlcA and Q3Rut in the range of 1 ng/mL to 25 μg/mL covering 10 different concentrations per decimal power. The linearity was estimated via the test of goodness of fit by Mandel before conducting the linear regression. The linearity, the limit of detection (LOD), and the limit of quantification (LOQ) were determined according to DIN 32645. Recovery and Repeatability. Ten samples with an analyte concentration of 5 or 1 μg/mL each were spiked with 3 μg of the internal standard Q3Rut, processed in parallel as described above, and analyzed via UHPLC−MS. The recovery was estimated indirectly by the accuracy and averaged over all measured samples after performing the outlier test by Nalimov. To ascertain the repeatability limit r, the series of measurements were considered separately. These parameters were determined according to DIN ISO 5725 as mentioned in the guideline for method validation of the German Federal Environmental Agency.27 Precision. The repeatability of the UHPLC−MS system and the method precision were determined by replicate analysis of spiked plasma samples and 10-fold measurement of the same calibration standards at concentrations of 1 and 5 μg/mL intraday and interday. Both are reported as their coefficient of variation (CV). The repeatability of the measuring instrument was determined according to ICES CM 1997/E:2 as mentioned in the guideline issued by the German Federal Environmental Agency,27 and the method precision was determined according to the aforementioned guideline.27 Robustness. The robustness of the method was established via 6fold measurement of the same sample during the batch over a time period of about 12 h. The trend test by Neumann was carried out, and the robustness was determined as the CV of the process stability. Evaluation was again conducted according to the German Federal Environmental Agency’s validation guideline.27 Statistics. All statistical calculations were carried out using Microsoft Excel and LaborValidate - Labormethoden-Validierung Version 2.8 (LABC - Labortechnik Müller and Zilger GbR, Hennef, Germany).

Article

RESULTS AND DISCUSSION

Q3GlcA was used as the only target analyte for method development and validation. Other quercetin-derived phase II metabolites, like quercetin-7-O-glucuronide or sulfated derivatives, are neither commercially available nor extractable from plant material. However, Q3glcA should provide chemical properties similar to those of other quercetin glucuronides, and therefore it has often been used as a reference substance.14,28 Although Q3GlcA is commonly used to quantify other phase II metabolites, binding affinity between HSA and metabolite, as well as the polarity of the analyte and pH of the plasma, are crucial for the extraction efficiency. Because of these facts, results for sulfated and methylated metabolites should be carefully interpreted. Effects of Solvents. In preliminary experiments, the influence of the solvents and buffers required for the dissolution of the enzymes was investigated with respect to the stability of the analyte. As PXIV needs to be dissolved in pure water (pH 7), this solvent was not included in these studies, as it corresponded to the previously performed control ensuring the stability of Q3GlcA. An external aqueous calibration was used for the determination of the recovery. From these experiments, it turned out that the solvent used for dissolution of pepsin, 0.1% of formic acid in water (pH 2), was the only one that did not affect the stability of Q3GlcA because the recovery was close to 100%. In contrast, losses of 40% were observed when the analyte was dissolved in a 5 mM solution of L-cysteine (pH 6.5) required by papain. L-Cysteine contains a terminal SH group, and quercetin and its derivatives are known to be able to react with proteins containing L-cysteine by derivatization of its thiol group.29 The weakly alkaline conditions caused by the urea solution needed for trypsin led to the complete loss of Q3GlcA. Flavonols, like quercetin derivatives, are known to autoxidize at their C-ring at pH ≥ 8, forming a benzofuranone intermediate before reacting to various more stable degradation products.30 Therefore, trypsin was not considered in the subsequent steps. The quercetin aglycone proved to be unstable under all conditions tested. Suitability of Enzymes and Optimization of Enzyme− Protein Ratios. Pepsin, papain, and PXIV were added to aqueous solutions of the analyte and to spiked plasma samples at ratios of 1:50, 1:500, and 1:1000 (w/w). The pH of each sample was adjusted to the respective optimum range of the enzymes, pepsin pH 2, papain pH 6.5, and PXIV pH 7. Regarding PXIV, the lower limit of the optimum range rather than the mean optimum of pH 8.8 was applied because the analyte showed significant instability under alkaline conditions. After incubation, the enzymes were inactivated thermally, and samples were filtered and transferred to the LC−MS without further steps of sample preparation. The recovery was determined by external calibration using an aqueous solution of Q3GlcA. Despite the low recovery observed when the Lcysteine solution was used, papain was included in these experiments to investigate whether the presence of the enzyme affects the stability of the analyte. From Figure 1, not showing the previous control, it can be seen that the presence of enzymes in the aqueous samples resulted in a decreased recovery as compared to the abovementioned results obtained when only the solvents were used. An exception was papain, which did not further affect the stability of the analyte. Addition of PXIV led to a loss of Q3GlcA of approximately 40%, which can be attributed 6667

DOI: 10.1021/acs.jafc.9b01782 J. Agric. Food Chem. 2019, 67, 6665−6671

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because the applied methodology without precipitation did not allow the analysis via LC−MS as the contained proteins would plug the chromatographic column. Inactivation of Enzymes and Protein Precipitation. To avoid plugging of the UHPLC columns by coagulating peptides and to minimize ion suppression during MS analysis, the matrix load needed to be reduced. It is known that matrix components may cause ion suppression via different mechanisms, such as coelution and competing for the available charge, bonding to the analyte, or even neutralization of analyte ions during gas-phase acid/base reactions.31 Therefore, precipitation using organic solvents combined with LLE rather than thermal treatment was performed to inactivate the enzyme and to remove peptides, as LLE proved to be a suitable method to separate some matrix components.31 For this purpose, different organic solvents were tested concerning their ability to precipitate the remaining peptides. Complete precipitation was accomplished by the following plasma− solvent ratios (v/v): acetonitrile 1 + 3, dimethylformamide 1 + 5, dimethyl sulfoxide 1 + 1, ethanol 1 + 3.5, methanol 1 + 2.5, and 2-propanol 1 + 5.5. These results are in agreement with previously described properties of the solvents.32 Even though dimethyl sulfoxide caused less dilution, it proved to be unsuitable for further use because it led to gel formation, and therefore the samples could not be analyzed via UHPLC−MS. All other solvents caused a more or less flocculent precipitate, which could easily be removed completely via centrifugation to obtain a clear supernatant. For these reasons, only these solvents were considered in the following method development. Evaporation to dryness led to poor recoveries, whereas recoveries of up to 84% were obtained when sample volumes were reduced (Table 1). On the basis of these results, only methanol was used as a precipitation agent in the last step of method development. Because no concentration of the analyte in the sample was achieved by this method, the process of evaporation had to be extended by reducing the residual volume to about one-half the initial plasma volume, resulting in a final analyte recovery of approximately 85% after methanolic precipitation. Validation. The validation was carried out on the basis of the Guidelines for Method Validation of the German Federal Environmental Agency.27 This allowed a more comprehensive description of the developed method with enhanced confidence level of the different parameters, because, contrary to the FDA’s Guidelines for Bioanalytical Method Validation,33 more samples had to be analyzed and more parameters had to be determined. During the validation, only matrix-matched calibration was used as exemplified before. The resulting calibration curve of Q3Rut was linear from 2.0 ng/mL to 17.5 μg/mL, and the calibration curve of Q3GlcA was linear from 1.5 ng/mL to 25 μg/mL, with a correlation coefficient (r2) greater than 0.996 in both cases. This linear range was wider than that found by Yang and co-workers,28 who reported 5 ng/ mL to 1 μg/mL for Q3GlcA. The linearity range should thus cover the working range necessary for samples derived from nutritional studies, as the reported concentrations ranged from 40 ng/mL to 3 μg/mL.14,34 The LOD of Q3GlcA was 2.0 ng/ mL, with the confidence interval (CI) being 1.4−3.4 ng/mL, and the LOQ was at 7.2 ng/mL, with the according CI ranging from 5.1 to 12.2 ng/mL. For the internal standard Q3Rut, the values were slightly higher, with a LOD at 3.7 ng/mL, with a CI of 2.6−6.4 ng/mL, and an LOQ of 13.5 ng/mL, with a CI

Figure 1. Influence of different enzyme doses on the recovery of Q3GlcA in aqueous samples and human plasma.

completely to the presence of the enzyme. According to the supplier, PXIV consists of a mixture of five serine-type proteases. Quercetin and its derivatives may bind to the specific substrate binding S1 pocket of serine-type proteases, causing an inhibition of the enzyme.19 Therefore, it is reasonable to assume that this binding may have caused the observed decrease in recovery. Furthermore, when lower enzyme−protein ratios were used, increased recoveries of 80− 90% were obtained during proteolysis with papain and PXIV. In contrast, this behavior was not observed for pepsin; the recovery remained rather constant at approximately 75% regardless of the enzyme−protein ratio. With respect to aqueous samples, PXIV and papain led to higher analyte recoveries than pepsin when used in lower ratios. Considering the results of the plasma samples, also shown in Figure 1, pepsin showed the best outcome with an analyte recovery of approximately 55% when a ratio of 1:1000 was used. In comparison, both papain and PXIV showed a 3-fold lower recovery. The quercetin-induced enzyme inhibition of PXIV19 may have caused an insufficient proteolysis and, thus, a minor release of the analyte. On the basis of these results, papain and PXIV were not considered further, and method development was performed exclusively with pepsin. For further optimization, lower enzyme−protein ratios (1:1000; 1:2500; 1:5000; 1:10 000) were tested, and the results are shown in Figure 2. Proteolysis in samples treated with the lowest amount of the enzyme was insufficient, leading to coagulation during the subsequent thermal treatment. Therefore, the analyte recovery could not be determined. A ratio of 1:5000 proved to be the best choice for further experiments. The performance of a positive control using untreated plasma would have been desirable but was impracticable

Figure 2. Development of the analyte recovery relating to reduced enzyme dosage. 6668

DOI: 10.1021/acs.jafc.9b01782 J. Agric. Food Chem. 2019, 67, 6665−6671

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Journal of Agricultural and Food Chemistry

Table 1. Comparison of Analyte Recoveries (%) after Organic Precipitation and Evaporation to Dryness with Dissolvation in 20% (v/v) Aqueous Methanol or to a Residual Volume of about 1000 μL with the Use of QRut as Internal Standarda acetonitrile

dimethylformamide

ethanol

methanol

2-propanol

Q3GlcA spiking (μg/mL)

dryness

remaining volume

dryness

remaining volume

dryness

remaining volume

dryness

remaining volume

dryness

remaining volume

1 5

31.0 ± 0.3 33.5 ± 0.6

n.t. 74.0 ± 19.1

16.9 ± 0.5 23.7 ± 7.3

n.t. n.t.

25.2 ± 7.9 10.6 ± 3.8

n.t. n.t.

30.5 ± 3.2 32.5 ± 1.7

n.t. 84.3 ± 10.1

7.9 ± 1.5 12.0 ± 0.9

n.t. n.t.

a

n.t. = not tested.

Table 2. Validation Parametersa analytical limits

Q3GlcA Q3Rut

precision

LOD

CI

LOQ

CI

system CV

method CV

recovery

repeatability

robustness

[ng/mL]

[ng/mL]

[ng/mL]

[ng/mL]

[%]

[%]

[%]

r

process stability CV [%]

2.0 3.7

1.4−3.4 2.6−6.4

7.2 13.5

5.1−12.2 9.4−23.6

1.7 2.9

24.2

67.3 ± 16.3

46.9 ± 16.7

3.6

a

LOD, limit of detection; LOQ, limit of quantification; CI, confidence interval; CV, coefficient of variation.

solubility, physicochemical stability, chemical reactivity, and ionization properties. The method presented by Wang and Morris is therefore qualified only for the analysis of quercetin as a sum parameter after enzymatic hydrolysis of the metabolites37 and not for analyzing the metabolites. In most cases, the quercetin metabolites were determined indirectly as their aglycone after enzymatic hydrolysis.34,38 However, this leads to a loss of information because the individual metabolism of the subjects cannot be determined. On the other hand, the determination as sum parameter allows a faster assessment of the analyzed samples, because fewer substances need to be identified and quantified. The respective linear range of the method of Wang and Morris is much smaller, only ranging from 1−200 ng/mL, but the LOD and LOQ are about 10 times lower than for the method presented here.37 The latter results might be caused by the use of a different ionization technique, turbo spray ionization (TSI) versus ESI, and a different MS detector, triple quadrupole versus linear ion trap,37 as these limits are known to be device-dependent.35 Yet, they are able to quantify the analyte with a higher precision of an interday CV of 11%, but only with an accuracy of 92−112%, and the recovery from plasma samples was not reported.37 Thus, although the method presented here showed poorer results regarding recovery and precision than the method of Yang and co-workers,28 or even the method of Wang and Morris to analyze the aglycone,37 it is still a viable and promising alternative for direct quantification of Q3GlcA in plasma samples. Some previously published methods used sample preparation methods for quantification of Q3GlcA that had been reported for identification but not quantification purposes.14,39 Thus, to our knowledge, the validation parameters of these methods have never been published,24 or the validation was insufficient as only the recovery was estimated23 without any further information about repeatability or precision. Information on these parameters is indispensable to know the resulting variance of the measured values from this method and thus to be able to evaluate its suitability and its quality. A mean value that might result from a wide scatter or from a large deviation within different measurement series is not sufficient to benchmark a method. In conclusion, enzymatic proteolysis proved to be a suitable method for the preparation of human plasma samples for

from 9.4 to 23.6 ng/mL. Further validation parameters are shown in Table 2. The validation data demonstrate that the UHPLC−MS system can reproducibly detect the analyte as well as the internal standard with high precision via the selected SRM mode, as the CV of the repeatability of the measuring instrument is 20% as LLOQ,28 but, in this case, the LLOQ was estimated as the lower limit of the CI of the LOQ. The interday method precision as well as the intraday method precisions with a CV of 23.7−26.3% are 15% are not uncommon regarding physiological matrixes. For example, comparable CVs are also found for some steroids.36 Although the average recovery determined is slightly lower than the minimum of 70% as required by the FDA,33 it is still satisfactory regarding the validation guidelines used and the different approach of estimation.27,35 The resulting low precision and recovery, as well as the comparatively high LOD and LOQ, might be caused by ion suppression, considering the fact that analyte and internal standard might be unequally affected.31 Via LLE, only few matrix components could be removed from the samples, whereas SPE is more efficient in reducing the matrix load.31 The recovery of the method reported by Yang and co-workers averaged 78.9% and was determined according to the guidelines of the FDA33 and not indirectly via accuracy as mentioned by the DIN ISO 5725. Regarding the determination of the robustness, the result confirmed that the samples were stable in the sample manager at 10 °C for at least 12 h. Another method for the analysis of quercetin metabolites in human plasma had been validated only for quercetin37 but not for its metabolites. Quercetin differs from its derivatives in its 6669

DOI: 10.1021/acs.jafc.9b01782 J. Agric. Food Chem. 2019, 67, 6665−6671

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Journal of Agricultural and Food Chemistry flavonol metabolite analysis via UHPLC−MS. However, it is unsuitable for the analysis of the quercetin aglycone. Therefore, it can be considered an alternative to the previously published methods like SPE and LLE of untreated plasma. In comparison to these conventional methods, the method presented here allows the release of noncovalently bound flavonol metabolites from plasma proteins through protein degradation before employing LLE or SPE. The enzymatic proteolysis is easy to handle and to reproduce in everyday laboratory work. Future adaptation to the analysis of other flavonoids seems conceivable. As most of the previously published methods used indirect quantification of quercetin metabolites in human plasma, this sample preparation method now allows a reliable and valid direct quantification of Q3GlcA in human plasma. The field of applicable methods such as SPE and LLE or even supported liquid extraction of pure plasma is now expanded by the introduction of enzymatic proteolysis prior to flavonoid metabolite analysis.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +49-228-73-4107. Fax: +49-228-73-4429. E-mail: [email protected]. ORCID

Maike Passon: 0000-0002-0002-2942 Andreas Schieber: 0000-0002-1082-9547 Funding

This research was supported by Diet Body Brain, Competence Cluster of Nutrition, funded by the German Federal Ministry of Education and Research (BMBF) (Grant no. 01EA1410A). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED CI, confidence interval; CV, coefficient of variation; HSA, human serum albumin; LLE, liquid−liquid extraction; LLOQ, lower limit of quantification; LOD, limit of detection; LOQ, limit of quantification; PXIV, protease type XIV from Streptomyces griseus; Q3GlcA, quercetin-3-O-glucuronide; Q3Rut, quercetin-3-O-rutinoside; SPE, solid-phase extraction; TSI, turbospray ionization

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