Determination of lipoic acid in human plasma by high-performance liquid chromatography with ultraviolet detection

2.1 Chemicals and reagents

1-Benzyl-2-chloropyridinium bromide (BCPB), a derivatization reagent, was synthesized in our laboratory as described earlier (Bald et al., 1996). Chloroform, hydrochloric acid, perchloric acid (PCA), sodium hydroxide and tris(hydroxymethyl)aminomethane (TRIS) were purchased from J.T. Baker (Deventer, the Netherlands). Acetic acid was purchased from Chempur (Piekary Śląskie, Poland). HPLC-grade acetonitrile (MeCN) and methanol (MeOH) were obtained from Labscan (Dublin, Ireland). Lipoic acid (LA), dihydrolipoic acid (DHLA), human serum albumin (HSA), 1-octanol and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) were purchased from Sigma–Aldrich (St. Louis, USA). Stock standard solutions were prepared by dissolving an appropriate amount of LA (final concentration 0.1 mol L⁻¹) in 0.2 mol L⁻¹ NaOH, BCPB (0.1 mol L⁻¹) and HSA (50 g L⁻¹) in water, and TCEP (0.25 mol L⁻¹) in TRIS buffer (pH 9). Stock solutions of LA and BCPB were kept at 4 °C for several days without a noticeable change of content. TCEP solution was prepared daily. The working solutions were prepared by dilution with water as needed.

2.2 Sample collection

Blood samples were donated by 17 apparently healthy, ethnically homogenous volunteers dosed with commercially available LA capsules (600 or 1200 mg of LA) for 30 days. During the study, no additional medications were allowed except LA. Blood (2 mL) was drawn, with a tourniquet applied, into a tube containing EDTA in the morning 1 day before the first LA dose and 1 day after last LA dose. Blood was centrifuged under standard conditions (10 min, 1000 g, within <20 min after collection) and plasma was used for the determination of LA. Informed consent was obtained from all volunteers, and this study was approved by the Bioethics Committee of Regional Chamber of Physicians in Krakow (127/KBL/OIL) and University of Łódź (22/KBBN-UŁ/II/2015). Plasma samples were stored at −78 °C until analysis. On the day of analysis, samples were thawed and processed as described below.

2.3 Preparation of Albumin-Cys³⁴-S-LA

HSA (50 g L⁻¹) dissolved in 0.1 mol L⁻¹ phosphate buffer pH 7.4 was converted to albumin-Cys³⁴-S-LA (HSA-LA) by incubation with 2-fold molar excess of DHLA or LA at 36.6 °C for 24 h. Excess LA was removed from HSA-LA by a quintuple extraction with chloroform as described in Section 2.5.2.

2.4 Separation of protein and LA

Portions of 50 μL of water or 50 g L⁻¹ HSA were each placed in eppendorf tube and spiked with 10 μL of standard solutions with the increasing amount of LA to provide a concentration in the range 0.1–20 μmol L⁻¹. Next, the mixtures were acidified with 7.5 μL of 3 mol L⁻¹ PCA, vortex-mixed and centrifuged at 12,000g for 5 min. The supernatant was transferred to new tubes and alkalized to pH 7 by the use of 2.5 mol L⁻¹ NaOH, 25 μL of 0.5 mol L⁻¹ TRIS buffer (pH 9) and 5 μL of 0.25 mol L⁻¹ TCEP were added and vortex-mixed. After 15 min, 10 μL of 0.1 mol L⁻¹ BCPB was added and the mixture was vigorously shaken by hand for 10 s. Next, the sample was kept at room temperature for 15 min and was acidified with 15 μL of 3 mol L⁻¹ PCA. The mixture was then transferred to an autosampler vial and injected (5 μL) into the HPLC system.

A 50 μL of water or 50 g L⁻¹ HSA was spiked with standard solution of LA (final concentration 10 μmol L⁻¹). Next, 25 μL of 0.5 mol L⁻¹ TRIS buffer (pH 9) and 5 μL of 0.25 mol L⁻¹ TCEP were added, vortex-mixed and allowed to stand for 15 min at room temperature. Next, 10 μL of 0.1 mol L⁻¹ BCPB was added, vigorously mixed, held on 15 min and acidified with 15 μL of 3 mol L⁻¹ PCA. At the end, different volume of MeCN (0–90 μL) or MeOH (0–80 μL) were added, vortex-mixed and put aside for 15 min, followed by centrifugation (5 min, 12,000g). Supernatant was transferred to an autosampler vial and injected (5 μL) into the HPLC system.

2.5 Recommended analytical procedure

2.6 Chromatographic analysis

The analyses were performed using a 1220 Infinity LC Agilent system consisted of a binary pump integrated with degasser, autosampler, column oven and diode-array detector. The whole HPLC system was controlled by OpenLAB CDS ChemStation Edition software. All separations were achieved through a Zorbax C-18 (150 × 4.6 mm, 5 μm) analytical column (Agilent Technologies). The mobile phase consisted of 2% acetic acid solution, pH 2.36 (component A) and MeCN (component B). For the determination of LA in plasma, gradient elution was used: 0–5 min, 10–40% B; 5–6 min, 40–10% B; 6–8 min, 10% B. Flow rate of mobile phase was 1 mL min⁻¹ at 25 °C. The peak of 2-S-pyridinium derivative of LA was monitored at 321 nm. LA-BCPB peak was identified by comparison of its retention time as well as diode-array spectra, taken at real time of analysis, with that of the authentic standard.

2.7 Validation study

3.1 Sample preparation

Sample preparation influences nearly all of the later assay steps and for this reason it is critical for unequivocal identification and quantification of the analytes. DHLA reacts with BCPB (Fig. 1) within 15 min in water solution (pH 9) to form a stable thioether, 2-S-pyridinium derivative (LA-BCPB). This stable derivative has a well-defined maximum at 321 nm. To measure the total LA (the sum of DHLA, LA and ProtS-LA) it is necessary to cleave disulfide bonds prior to derivatization. Such approach makes free sulfhydryl groups accessible to BCPB. For this reason, we used TCEP in TRIS buffer at pH 9, at room temperature for 15 min. Our results show that both reduction and derivatization require longer times for plasma samples than for urine (Chwatko et al., 2014). A crucial step in plasma sample preparation is LA and DHLA separation from protein. LA and DHLA are the amphiphilic molecules having both hydrophilic and hydrophobic fragments and can specifically interact with the surface of proteins. Several methods, such as liquid-liquid extraction (LLE) (Chng et al., 2010; Ezhilarasi et al., 2014; Haj-Yehia et al., 2000; Khan et al., 2011a, 2010, 2011b; Montero et al., 2012; Niebch et al., 1997; Satoh et al., 2007; Trivedi et al., 2004; Witt and Rüstow, 1998), solid phase extraction (Khan et al., 2010; Teichert and Preiss, 2002, 1995) and deproteinization (Chen et al., 2005; Chng et al., 2010; Khan et al., 2011b) can be utilized for removal of LA from protein. In our study, we tested deproteinization procedure and LLE for determination of total LA and ProtS-LA, respectively.

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Figure 1 Determination strategy for estimation of total and protein-bound LA in plasma.

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During acidic deproteinization, plasma proteins markedly adsorb LA; consequently, the concentration of LA in the solution is lower than expected. In our study, HSA solutions containing LA in the range 0.5–30 μmol L⁻¹ were deproteinized with PCA and assayed for LA content. We observed a lower LA concentration in final analytical solutions for HSA samples compared to standard solution (Fig. 2A). Recovery of free LA was calculated with the use of the following formula: $$\text{Recovery}(\%)=\text{LA amount in HSA sample}/\text{LA amount in standard solution}\times 100\%\text{.}$$

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Figure 2 Influence of different conditions to separation of LA and LA-BCPB from protein. (A) Acid deproteinization of HSA containing LA in the range 0.5–20 μmol L⁻¹; (B) The relationship between MeCN/MeOH volume and separation of LA-BCPB from protein.

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The recovery was found to be between 32.4% and 38.9%, meaning that more than 60% of free LA adsorbed on HSA. Similar problem concerned separation of proteins and BCPB derivative of LA. Our experiments have proven that addition of MeCN or MeOH to PCA significantly increases LA-BCPB recovery (Fig. 2B). Moreover, better results were obtained when the mixture of PCA and MeCN was used. In this case, the recovery reaches 100% while application of MeOH and PCA mixture afforded only 76%. Our results are comparable with those obtained by other investigators for the mixture of MeCN and metaphosphoric acid (recovery, 98%) (Khan et al., 2011b). Finally, for deproteinization of plasma samples for determination of total LA the mixture of PCA and MeCN was used.

Since LA shares structural similarity to the medium chain fatty acids e.g. octanoic acid it is preferably bounded by sit II in albumin (Atukeren et al., 2010; Kawakami et al., 2006). An accurate separation of free LA from proteins is essential for determination of ProtS-LA in plasma samples because incomplete removal of free LA causes overestimation of ProtS-LA. Thus, LLE with chloroform, dichloromethane, diethyl ether, ethyl acetate or 1-octanol was considered. As described earlier, dichloromethane, diethyl ether and ethyl acetate were widely used for LA extraction from plasma. Unfortunately, one can interpret provided results as inconsistent due to the extraction recoveries in case of dichloromethane, diethyl ether and ethyl acetate being in the range of 87.0–98.4%, 75.0–80.3% and 58.1–90.3%, respectively (Haj-Yehia et al., 2000; Montero et al., 2012; Khan et al., 2011b, 2011a, 2010; Trivedi et al., 2004; Witt and Rüstow, 1998). Additionally, when chloroform/ethanol mixture was used for LA extraction from plasma samples better recovery of the analyte was obtained as compared to the other solvents used (Montero et al., 2012). Then, we have decided to test chloroform and 1-octanol as the extraction solvents. Our results show that both solvents completely extracted free LA from acidified HSA and plasma samples. Additionally, after extraction of HSA containing free LA (10 μmol L⁻¹) the LA-BCBP peak in protein fraction was not observed. Due to the better stability of proteins as well as better separation from the liquid phases for further experiments chloroform was used.

3.2 Chromatography

Because of unique separation parameters of HPLC technique, it has frequently been used in the analysis of biological samples. In order to provide a good performance of the assay the chromatographic conditions were optimized in terms of mobile phase MeCN and acetic acid concentration as well as column temperature. As expected, an increase in MeCN content in the mobile phase from 20 to 30% resulted in decrease of retention factor from 8 to 1 and simultaneous increase in peak height from 6 to 32 mAU. The effect of acetic acid concentration was tested in a range of 0.5–3%. The retention factor of LA-BCBP slightly decreased (1.4–1) while peak height increased (16–40 mAU) with increase of acetic acid concentration. Temperature of the column in the range of 15–40 °C slightly influenced retention parameters. Based on the results described above, we have chosen chromatographic conditions specified in Section 2.6. Chromatographic profiles obtained for the standard, blank and spiked protein fraction as well as total LA after supplementation are depicted in Fig. 3. As can be seen endogenous components do not significantly interfere with LA-BCBP peak. Under these conditions LA-BCPB is eluted after 5.49 ± 0.01 min (RSD = 0.18%, n = 10).

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Figure 3 Representative chromatograms of water standard solution and plasma samples. (A) Standard water solution of LA, concentration 15 μmol L⁻¹. (B) Profile of plasma obtained from volunteer before (black line) and after oral administration of 600 mg (red line) and 1200 mg (blue line) of LA with total LA amounting 0.0 μmol L⁻¹, 0.53 μmol L⁻¹ (108.3 μg L⁻¹) and 2.08 μmol L⁻¹ (430.0 μg L⁻¹), respectively. (C) Profile of blank (black line) and spiked (red line) with 3 μmol L⁻¹ ProtS-LA plasma samples. Chromatographic conditions are as described in the text. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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3.3 Validation study

The method was validated for selectivity, precision, recovery, linearity, limit of detection and quantitation, and stability according to the guidelines for analytical methods (Ravichandran et al., 2010; Wille et al., 2011).

The selectivity of our method was demonstrated by providing the absence of interfering peaks with a signal obtained for the analyte. For this purpose, six various blank plasma samples and plasma spiked with LA were analyzed and the chromatograms showed sufficient separation of LA-BCPB from the matrix components. Furthermore, examination of peak purity showed that LA-BCBP peak was not attributable to more than one component.

The accuracy of an analytical method may be defined as the closeness of the measured value obtained by the method to the true value, whereas the precision as the degree of agreement among individual test results obtained by replicate measurements (Ravichandran et al., 2010; Wille et al., 2011). The precision is expressed as the relative standard deviation (RSD), while the accuracy as the percentage of analyte recovery and calculated by expressing the mean measured amount as a percentage of the added amount. In our study three concentrations of LA were tested: one at the LOQ, one near the expected amount of analyte, and one near the upper boundary of the standard curve. The intra- and inter-day recovery and precision values were calculated and are shown in Table 1. The criteria for acceptability of the data included accuracy within ±15% from the nominal values and a precision within ±15% RSD, except for LOQ, where it should not exceed ±20% for accuracy as well as precision (Wille et al., 2011). Obtained in our study values fulfill these requirements.

The relationship between detector response and LA and ProtS-LA concentrations was continuous and repeatable, and was demonstrated using a seven-point and six-point calibration curves, respectively. The calibration curves were linear in the tested range from 0.1 to 20 μmol L⁻¹ and from 0.5 to 3 μmol L⁻¹ for total LA and ProtS-LA, respectively. The equations for the linear regression line were y = 1.295x + 0.0726 for total LA and y = 0.330x − 0.018 for ProtS-LA; outliers were not excluded. The coefficients of correlation for the calibration regression were 0.9998 and 0.9901 for total LA and ProtS-LA, respectively.

The LOD and LOQ for total LA and ProtS-LA were experimentally established to be 0.05 and 0.1 μmol L⁻¹ for total LA and 0.2 and 0.5 μmol L⁻¹ for ProtS-LA, respectively. The analyte response at LOQ level fulfills the criteria for precision and accuracy (Wille et al., 2011), i.e. precision of 20% and accuracy of 80–120%. The LOQ and/or LOD for total LA determination by this method compare favorably with others’ recently reported values which were obtained by the HPLC method with UV detection (Ezhilarasi et al., 2014) and match with the HPLC method with fluorescence detection (Niebch et al., 1997). Other HPLC methods for LA determination in plasma based on fluorescence (Haj-Yehia et al., 2000; Satoh et al., 2007; Witt and Rüstow, 1998), mass spectrometry (Chen et al., 2005; Trivedi et al., 2004) and electrochemical (Khan et al., 2011a, 2010, 2011b; Teichert and Preiss, 2002, 1995) detection have better LOQ and/or LOD than our method.

The stability of LA ex vivo at 24, 4, −20 and −78 °C in plasma was tested. Under these temperatures LA in plasma samples was found to be stable for at least 2, 10 days, 26 weeks and 6 months, respectively. The residual percentages of LA after mentioned periods of time were 96.5%, 102.5%, 98.7% and 103.0%, respectively and indicating no stability problems. Our stability results correspond to those obtained earlier at 24 (Khan et al., 2011b), −20 (Khan et al., 2011a; Chen et al., 2005) and −80 °C (Trivedi et al., 2004) and are inconsistent with the study provided by Khan et al. (2010) which demonstrated that spiked plasma samples were stable for only 1 week at −80 °C. Other examinations have shown that exogenous LA in plasma samples is stable at −70 °C for 6 months (Teichert and Preiss, 1995). LA-BCPB in final analytical solution containing MeCN was found to be stable at room temperature for 4 h as the concentration of analyte was found to be 103.6% of the initial concentration.

3.4 Application to authentic plasma samples

The optimized procedure for the determination of total LA and ProtS-LA was applied to the analysis of plasma samples of apparently healthy volunteers, 28–63 years old (7 men and 10 women), before and after supplementation. In 14 of the 17 plasma samples collected before taking the drug the LA levels were below the LOD and in 3 samples below the LOQ. After oral administration of 600 and 1200 mg of LA, its concentration in plasma was in the range 33.9–287.8 and 260.7–430.0 μg L⁻¹ plasma, respectively (Table 2). Furthermore, in all plasma samples ProtS-LA was not observed.