Notes
Determination of Miglitol by Column-Switching Ion-Pair HPLC with Tris(2,2′-bipyridine)ruthenium(II)-Electrogenerated Chemiluminescence Detection
2015 Volume 63 Issue 6 Pages 476-480
Details
2015 Volume 63 Issue 6 Pages 476-480
We have developed a highly sensitive, simple method for the quantitative determination of miglitol in standard serum samples using column-switching ion-pair HPLC with tris(2,2′-bipyridine)ruthenium(II)-electrogenerated chemiluminescence detection. The serum samples were directly injected into a column-switching HPLC system with a Shim-pack MAYI-SCX precolumn to remove the serum matrix. Chromatographic separation of miglitol was achieved on a TSKgel ODS 100-V column using a mobile phase containing sodium 1-octanesulfonate as an ion-pair reagent. The detection and quantification limits of miglitol were 3 and 10 ng/mL, respectively. The calibration curve for miglitol in the serum samples showed good linearity (r2=0.9997) in the range of 10–2500 ng/mL. The recovery rate of miglitol from the serum samples was more than 94% as calculated from blank serum samples spiked with miglitol 50, 100, 500, 1000, and 2000 ng/mL. Therefore, this method can be applied to routine therapeutic monitoring of miglitol in serum samples.
Type 2 diabetes, formerly called non-insulin-dependent diabetes, is a costly disease; in 2013, approximately 300 million people worldwide were affected, according to the International Diabetes Federation.1) Miglitol, a type of α-glucosidase inhibitor (α-GI), is useful for the treatment of type 2 diabetes because it inhibits the activity of disaccharide-hydrolyzing enzyme in the small intestine, which has a lowering effect on postprandial blood glucose and insulin levels.2–5) Recently, it was demonstrated that miglitol prevents reactive hypoglycemia secondary to late dumping syndrome. Furthermore, miglitol is more effective than two other α-GIs, voglibose and acarbose.6) To achieve rapid therapeutic monitoring and pharmacokinetic studies of miglitol, a rapid and simple method for its quantification in blood samples is required. The enzyme inhibition assay for quantification of miglitol in plasma and urine samples seems unreliable with regard to sensitivity and selectivity.7) An alternative is the liquid chromatographic-tandem mass spectrometry (LC-MS/MS) assay for the quantification of miglitol in plasma,8,9) which is sensitive and can give reliable results, but requires expensive instrumentation and has a high operating cost. In addition, this method requires a time-consuming cleanup procedure to remove interference in biological samples. Thus, it is not suitable for routine analysis of miglitol.
We previously reported the development and application of a column-switching HPLC method with tris(2,2′-bipyridine)ruthenium(II)-electrogenerated chemiluminescence (ECL) detection.10–14) Chemiluminescence (CL) has become a powerful analytical tool for a large variety of analyses because it has a low detection limit and wide linear dynamic range while requiring relatively simple instrumentation. The column-switching system avoids off-line sample preparation procedures such as traditional solid-phase extraction or liquid–liquid extraction.15–19) This technique allows plasma samples to be directly injected onto the HPLC system, thereby resulting in an accurate assay without internal standards. As shown in Chart 1, detection using tris(2,2-bipyridine)ruthenium(III) (Ru(bpy)33+) has been shown to be sensitive and selective for reducing agents such as indoles,20) oxalate,21) active methylenes,22) and tertiary amines.23) Miglitol contains a tertiary amine; therefore, Ru(bpy)32+ ECL is effective in its detection.
The detection system has the advantages of having no derivatization step and a simple sample preparation procedure due to its high sensitivity and selectivity. As shown in Fig. 1, miglitol is structurally a glucose analog, which is not easy to detect due to the lack of a chromophore or fluorophore in its chemical structure. Furthermore, miglitol is a hydrophilic compound. Water-rich mobile phases are widely used in chemiluminescence detection; therefore, reversed-phase columns must be used here. An ion-pair chromatographic system, equipped with an octadecylsilyl (ODS) column and a mobile phase containing sodium 1-octanesulfonate as the ion-pair reagent, was used in this study.
The purpose of this study was to develop and validate a rapid and selective method for the quantification of miglitol in serum samples by column-switching ion-pair HPLC with Ru(bpy)32+ ECL. This method is simple compared with other methods, and does not require special sample preparation.
Miglitol was obtained from Sanwa Kagaku Kenkyusho Co., Ltd. (Nagoya, Japan). This material is soluble in water and has a pKa24) of 5.9. Moreover, it has very low Log P25) value (−2.3). Tris(2,2′-bipyridine)ruthenium(II) chloride hexahydrate (Ru(bpy)3Cl2·6H2O) was purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). Sodium dihydrogen phosphate, acetonitrile, and the liquid control serum I were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sodium 1-octanesulfonate was obtained from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Water was purified with a Still Ace SA-2000E (Tokyo Rikakikai Co., Ltd., Tokyo, Japan). A standard stock solution of miglitol was prepared in water and stored at 4°C. Working solutions were prepared by appropriately diluting the stock solution before use. A 10 µL aliquot of the diluted miglitol solution was mixed with a 990 µL of the liquid control serum I and the sample (20 µL) was injected into the column-switching HPLC.
Column-Switching HPLC ConditionsFigure 2 shows a schematic diagram of the column-switching ion-pair HPLC with ECL detection system. Eluent-1 (E-1) was 2 mM sodium dihydrogen phosphate buffer (pH 4.0)–acetonitrile (94 : 6, v/v) and Eluent-2 (E-2) was 80 mM sodium dihydrogen phosphate buffer (pH 5.0) containing 10 mM sodium 1-octanesulfonate. The flow rate of E-1 was 1.2 mL/min, controlled with a LC-6A HPLC system (Shimadzu, Kyoto, Japan) equipped with a 7125 sample injector (20 µL; Rheodyne, Cototi, CA, U.S.A.); flow rate for E-2 was 1.0 mL/min, controlled with a uf-3005SZB2 pump (Uniflows, Tokyo, Japan). The CL reagent solution for the post-column reaction was Ru(bpy)32+ in 2.5 mM sulfuric acid solution, which was delivered at a flow rate of 0.3 mL/min with an Intelligent Pump 301 (Flow, Tokyo, Japan). The concentration of Ru(bpy)32+ solution, 0.3 mM was chosen as optimal, following previously described methods.10–14)
E-1, E-2, and reagent solution were pumped through a DG-2410 (Uniflows) degasser. The six-port switching valve was made by Kyowa Precision Industries (Tokyo, Japan). A Shim-pack MAYI-SCX (10×4.6 mm i.d., Shimadzu) was used as the precolumn (C-1) and a TSKgel ODS 100-V (150×4.6 mm i.d., 5 µm, Tosoh Co., Ltd., Tokyo, Japan) was used as the analytical column (C-2). C-1 and C-2 were used at room temperature. Ru(bpy)33+ is unstable in aqueous solution, so was prepared from Ru(bpy)32+ before use. The Ru(bpy)32+ solution was delivered and oxidized to Ru(bpy)33+ by the controlled-current electrolysis method (Galvanostat Comet 2000, Comet, Kawasaki, Japan). The electrolytic current of the electrochemical reactor was set at 80 µA. A Pulse Dumper (Uniflows) was placed between the pump and the electrode cell to prevent pulsation due to the pump. The E-2 and Ru(bpy)33+ solutions were mixed and pumped continuously through the spiral cell in the detector. Chromatograms were recorded with a chromatopac C-R5A processor (Shimadzu).
Column-Switching ProcedureThe system was operated according to the following procedure, with six port valve positions and switchover times given in parentheses. Step 1 (position A; 0–5 min): A serum sample was injected onto C-1, which was washed with E-1 in order to remove serum proteins and other endogenous interferences. Step 2 (position B; 5–6 min): The valve was switched from position A to position B; miglitol was eluted from C-1 to C-2 in the back-flush mode. Step 3 (position A; 6 min): The valve position was returned to the initial condition and miglitol separated in C-2. Since a recovery rate (%) of miglitol in this system was in a gradual decline when the number of serum sample injection was over 150, C-1 was replaced after injection of at most 150 serum samples.
Validation of HPLC AnalysisTo validate the analytical method, the lower limits of quantification, linearity, precision, and recovery rate of miglitol in serum samples were evaluated. The linearity of the method was examined by spiking control serum with a standard solution of miglitol: a calibration curve was obtained by plotting peak areas versus known concentrations. The coefficient of determination (r2) was determined. The lower limit of quantification was the smallest analytical concentration of miglitol with a coefficient of variation of less than 20%. The intra-day and inter-day precision (coefficient of variation, CV) was estimated by analyzing five replicates containing miglitol at six different concentrations: 20, 50, 100, 500, 1000, and 2000 ng/mL. The acceptance criteria for intra-day and inter-day validation are below 10% bias. The recovery rate was determined by injecting a standard solution of miglitol onto the system and comparing its peak area with those produced by serum spiked with miglitol.
Adequate chromatographic separation was obtained using the single-column method, in which the precolumn was removed from the column-switching system. Miglitol is a highly polar compound presenting weak interactions with the reversed-phase stationary phase. In order to enhance method selectivity and retention factor (k′) of miglitol, an ion-pair reagent, sodium 1-octanesulfonate, was added to the mobile phase. First, the effect of sodium 1-octanesulfonate in the concentration range 5−15 mM was studied. Figure 3 shows that an increase in sodium 1-octanesulfonate concentration resulted in an increase in retention time, which is unfavorable for routine analysis, and had no influence on background noise. Moreover, there is fear that an increase in sodium 1-octanesulfonate concentration resulted in a deterioration of the column. In order to achieve an acceptable retention time, 10 mM concentration was chosen.
The Ru(bpy)32+ CL reaction is well known to be dependent on pH. The carrier solution was 80 mM phosphate buffer containing 10 mM sodium 1-octanesulfonate buffer (pH 3.0–6.0), and the reagent solution was 0.3 mM Ru(bpy)32+ in 2.5 mM H2SO4. The two solutions were pumped at flow rates of 1.0 mL/min and 0.3 mL/min, respectively. Figure 4 shows that the CL signal increased with increasing pH, but that values higher than pH 5.5 are not recommended because background noise increases concomitantly: background emission is caused by the interaction between Ru(bpy)33+ and hydroxide ion. In addition, when the buffer pH was higher than pH 5.0, retention factor decreased. Actually, when the buffer pH was higher than pH 5.5, the peaks of miglitol and foreign substances in serum were not separated in a chromatogram (data not shown). Consequently, pH 5.0 was chosen as optimal.
To remove endogenous interference, we employed the column-switching method instead of off-line sample preparation. Miglitol is a highly polar and basic compound; thus, a cation-exchange precolumn was employed. We evaluated the influence of the co-ion concentration (sodium ion), buffer pH range, and organic solvent (acetonitrile) concentration in the extraction mobile phase on the recovery rate of miglitol in this column-switching system. The retention factor of miglitol decreased gradually with rising pH in the phosphate buffer and increasing co-ion concentration: the retention mechanism of the precolumn is based on cation exchange. Two milli-molar phosphate (pH 4.0) was adopted as optimal. Complete exclusion of proteins was achieved within 5 min. The addition of 1–6% of organic solvent improved sample cleanup by avoiding undesired adsorption of endogenous substances on the surface of the hydrophobic phase: 6% organic solvent removed endogenous interference altogether. Thus, endogenous interference from serum samples was excluded within 5 min at a flow rate of 1.2 mL/min using 2 mM phosphate buffer (pH 4.0)–acetonitrile (94 : 6, v/v).
The extraction recovery rate of a standard sample from the column-switching system was calculated by directly comparing the peak heights from the analytical column (the single-column system); i.e., the precolumn was removed from the system. Table 1 shows recovery rate to be approximately 100%. The effect of transfer time from C-1 to C-2 is shown in Fig. 5. Using a 1 min transfer time, miglitol eluted from C-1 could be completely introduced to C-2. This short transfer time also limited the transfer of interfering compounds. Moreover, miglitol was not detected in the subsequent purified water injection for washing the system, indicating that there was no carry-over detected in the system. The column-switching technique provides high reproducibility and a rapid sample preparation procedure.
| Standard solution | Peak area/µV s | |||
|---|---|---|---|---|
| Single column | Column switching | |||
| Measured | Mean | Measured | Mean | |
| Miglitol (1 µg/mL) | 264700 | 265900 | 263600 | 262900 |
| 263600 | 262900 | |||
| 269500 | 262100 | |||
Typical chromatograms obtained from samples of blank serum (A) and serum spiked with miglitol (B) are shown in Fig. 6. Under optimum conditions, the retention time of miglitol was approximately 13.3 min, and miglitol was completely separated from serum endogenous materials. The detection limit for miglitol was 3.0 ng/mL at a signal : noise ratio of 3 : 1 and the lower limit of quantification was 10 ng/mL at a signal : noise ratio of 10 : 1. The calibration curve showed good linearity in the range 10–2500 ng/mL, and r2 was greater than 0.9997. In the LC-MS/MS method described above,9) the detection limit for miglitol was 1.0 ng/mL and the lower limit of quantification was 5.0 ng/mL. Thus, it was demonstrated that the ECL Ru(bpy)32+ detection method have a high sensitivity comparable to the LC-MS/MS method. On the other hand, although a deproteination with acetonitrile and a washing with dichloromethane of a plasma sample were required in the LC-MS/MS method, our method not requires a time-consuming cleanup procedure to remove interference in serum samples. Therefore, the ECL Ru(bpy)32+ detection method can be applied to routine therapeutic monitoring of miglitol in serum samples. As previously reported,7) miglitol is virtually not bound to plasma proteins. Actually, in this work, the recovery rate of miglitol from control serum was 94.3−98.5% (Table 2). Furthermore, CV for intra-day variation was in the range 2.52–8.37%, and for inter-day variation 0.97–9.87% (Table 3). These results indicate that this is a suitable method for analyzing control serum samples. In all instances, the precision, and recovery rate were satisfactory.
(A) Blank serum; (B) Serum spiked with 500 ng/mL of miglitol.
| Concentration added (ng/mL) | Concentration found (ng/mL) | Recovery (%) |
|---|---|---|
| 50 | 47.2±2.18 | 94.3 |
| 100 | 98.5±5.11 | 98.5 |
| 500 | 486.0±19.4 | 97.2 |
| 1000 | 984.0±29.0 | 98.5 |
| 2000 | 1964.0±49.5 | 98.2 |
| Concentration (ng/mL) | Intra-day (n=5) | Inter-day (n=3) |
|---|---|---|
| CV (%) | CV (%) | |
| 20 | 8.37 | 9.87 |
| 50 | 4.62 | 5.04 |
| 100 | 4.52 | 6.20 |
| 500 | 3.99 | 4.71 |
| 1000 | 2.63 | 1.05 |
| 2000 | 2.52 | 0.97 |
We developed a column-switching ion-pair HPLC method with ECL Ru(bpy)32+ detection for determining miglitol concentration directly in serum samples. The miglitol was detected within 18 min using a column-switching system with a precolumn for extraction and deproteinization and an analytical column for complete separation. The calibration curve for miglitol in the samples showed good linearity (r2=0.9997). Moreover, the detection limit of miglitol was 3.0 ng/mL. Therefore, this sensitive method can be applied to routine therapeutic monitoring of miglitol in serum samples.
The authors declare no conflict of interest.
