Abstract
New, simple, selective, and sensitive liquid chromatography–ultraviolet (LC–UV) methods have been developed and subsequently validated for simultaneous determination of linagliptin–empagliflozin combination and simultaneous determination of alogliptin benzoate–pioglitazone hydrochloride combination. Linearity was found to be acceptable over the concentration ranges of 2–50 μg mL−1, 4–100 μg mL−1, 0.5–25 μg mL−1, and 1–25 μg mL−1 for linagliptin (LNG), empagliflozin (EMG), alogliptin (ALG), and pioglitazone (PGN), respectively. All the methods were applied successfully to the analysis of the pharmaceutical dosage forms. The optimized methods were validated and proved to be robust and accurate for the quality control of the mentioned drugs in their different pharmaceutical dosage forms.
1. Introduction
Empagliflozin (EMG) (Figure 1a) is an inhibitor of sodium glucose co-transporter-2 (SGLT-2), so it inhibits glucose re-absorption into the blood [1]. Linagliptin (LNG) (Figure 1b) and alogliptin (ALG) (Figure 1c) are inhibitors to dipeptidyl peptidase-4 (DPP-4), so they stimulate insulin release [2]. Pioglitazone (PGN) (Figure 1d) is one of the thiazolidinediones which increase insulin sensitivity [3].
Chemical structures of LNG (a), ALG (b), EMG (c), and PGN (d)
Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2016.00089
To the best of the authors' knowledge, one chromatographic method [4] and one spectrophotometric Vierodt's method [5] were described for simultaneous determination of EMG and LNG. Furthermore, simultaneous equation and partial least square 2 (PLS-2) methods were used for simultaneous determination of EMG and metformin [6]. Moreover, the literature showed few chromatographic methods [7–10] and one spectrophotometric method for simultaneous determination of ALG and PGN based on first derivative and dual wavelength techniques [11].
Two new proposed methods have been discussed in the present work. The aim of the developed high-performance liquid chromatography (HPLC) methods is to present a new simple HPLC method for simultaneous determination of LNG and EMG in bulk powder, in their different ratios as laboratory-prepared mixtures and in their pharmaceutical dosage form (HPLC-1), and to present another accurate and precise HPLC method for simultaneous determination of ALG and PGN in bulk powder and in their pharmaceutical dosage form (HPLC-2).
The new methods were developed without use of any buffer applying simple mobile phases with the advantage of easy preparation and enhanced resolution results and offering mobile phases that are suitable for both UV detection and mass spectrometric detection. The present work was proved to be more economic than most of the developed methods in the literature [4–11] as lower retention times ensures lower run time that decrease the required amount of mobile phase. Furthermore, the calculated resolution values from the present investigation showed satisfactory results that ensures good separation and accurate determination of the drugs.
2. Experimental
2.1 Instrumentation.
The HPLC (10 AD, Shimadzu, Japan) consisted of a system controller (SCL-10A), degasser (DGU-12A), and UV–visible detector (SPD-10A) using XTERRA® C18 column (250 mm × 4.6 mm, 5 μm), and software class VP and Elmasonic S 60 H degasser (Germany) were used.
2.2 Reagents and Reference Samples.
EMG and LNG were found to be 99.71% and 100.21%, respectively, using reference method [4]; Glyxambi® tablets nominally containing 5 mg (LNG) and 10 mg (EMG) per tablet were supplied from Boehringer Ingelheim pharmaceutical company (Germany). ALG and PGN were found to be 99.87% and 100.03%, respectively, using reference method [10]; Oseni® tablets nominally containing 34 mg of alogliptin benzoate equivalent to 25 mg ALG and 33.06 mg of pioglitazone hydrochloride equivalent to 30 mg PGN per tablet were supplied from Takeda Pharmaceutical Company (Japan). HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific (Loughborough, Leicestershire, UK). Orthophosphoric acid (85%) was purchased from VWR Chemicals (Pool, England). Formic acid was purchased from Sigma-Aldrich (Deisenhofen, Germany).
2.3 Standard Stock Solutions.
Standard stock solutions of LNG, EMG, ALG, and PGN (1 mg mL−1) were prepared separately in methanol.
2.4 Working Solutions.
All the working solutions were prepared separately using the mobile phase of each experiment. Solutions of LNG (100 μg mL−1), EMG (200 μg mL−1), ALG (50 μg mL−1), and PGN (50 μg mL−1) were prepared.
2.5 Chromatographic Conditions.
All the mobile phases were filtered through 0.2-μm membrane filter and degassed for 30 min in an ultrasonic bath prior to its use. The column temperature was kept at 25 °C, and the injection volume was 20 μL. For HPLC-1, 0.1% aqueous formic acid–methanol–acetonitrile (40:20:40, v/v/v), pH 3.6, was used as a mobile phase at a flow rate of 2 mL min−1 with UV detection at 226 nm. For HPLC-2, mobile phase consisting of 50% methanol at pH 2.7 (adjusted with orthophosphoric acid) was used at a flow rate of 0.8 mL min−1 with UV detection at 270 nm.
2.6 Sample Preparation.
Twenty tablets of Glyxambi® and Oseni® were separately weighed, powdered, and mixed in a mortar. Separately, accurately weighed amounts of the finely powdered Glyxambi® and Oseni® tablets equivalent to 20 mg LNG and 40 mg EMG and 10 mg ALG and 9.7 mg PGN, respectively, were made up to 100 mL with methanol and sonicated to dissolve; then, the solutions were filtered.
2.7 Procedure and Method Validation
2.7.1 Linearity.
Linearity was achieved over the concentration ranges of 2–50 μg mL−1, 4–100 μg mL−1, 0.5–25 μg mL−1, and 1–25 μg mL−1 for LNG, EMG, ALG, and PGN, respectively, using LC–UV. Calibration curves were obtained by plotting peak area of six calibrators against concentration, and the regression equations were calculated.
2.7.2 Assay of ALG, PGN, LNG, and EMG in bulk.
Concentrations equivalent to 5, 15, 25, 35, and 45 μg mL−1 of LNG; 10, 30, 50, 70, and 90 μg mL−1 of EMG; 2.5, 7.5, 12.5, 17.5, and 22.5 μg mL−1 of ALG; and 2.5, 7.5, 12.5, 17.5, and 22.5 μg mL−1 of PGN were prepared, and all the concentrations were calculated using regression equations.
2.7.3 Precision.
Concentrations equivalent to 20, 25, and 30 μg mL−1 LNG; 40, 50, and 60 μg mL−1 EMG; and 8, 10, and 12 μg mL−1 of both ALG and PGN were prepared and analyzed three times within the same day and on three successive days. The mean % recoveries and the relative standard deviations were calculated.
2.7.4 Assay of LNG, EMG, ALG, and PGN in laboratory-prepared mixtures.
Different ratios (1:5, 2:5, 3:5,…..5:1) of LNG–EMG and ALG–PGN binary mixtures were prepared. Their concentrations were calculated using regression equations.
2.7.5 Robustness.
Flow rate was changed by ±0.1, the organic strength was changed by ±2%, and pH value of the mobile phase was varied by ±0.1.
2.7.6 Assay of ALG, PGN, LNG, and EMG in Glyxambi® tablets and Oseni® tablets.
The sample solutions prepared under section Sample Preparation were diluted, and then, the procedures mentioned above were adopted. The concentrations of the mentioned drugs were calculated using their regression equations. Then, to check the validity of the proposed method, the standard addition technique was applied by adding different known concentrations of the pure drug to fixed concentrations of the drug product.
2.7.7 Limit of detection and limit of quantitation.
Limit of detection (LOD = 3.3 × residual standard deviation of regression line / slope) and limit of quantitation (LOQ = 10 × residual standard deviation of regression line / slope) were determined for the proposed methods.
3. Results and Discussion
The proposed methods proved to be simple, accurate, and reproducible for the determination of LNG, EMG, ALG, and PGN, and they were validated showing satisfactory data for all the parameters tested. The new methods were developed without buffer with the advantage of easy preparation and enhanced resolution results and offering mobile phases that are suitable for both UV detection and mass detection.
3.1 Method Development.
According to the literature review regarding HPLC analysis of gliptins (LNG and ALG), Cyano column was used for chromatographic determination of single gliptins [12] while C18 column was used successfully for simultaneous determination of gliptins and other drugs in combination [13] with sharp peaks and good resolution, so C18 column was selected for the present work to ensure satisfactory separation of LNG–EMG peaks and ALG–PGN peaks.
For the first HPLC method, using both acetonitrile and methanol was crucial to enhance the resolution between LNG and EMG, and also, using 0.1% formic acid was enough to adjust the acidity of the mobile phase to pH 3.6. Flow rate was adjusted to be 2 mL min−1 to decrease the retention time of the studied drugs without affecting the resolution between peaks. Finally, UV detection at 226 nm was the most sensitive wavelength for the determination of the two drugs simultaneously. Retention times for LNG and EMG were found to be 1.5 and 3 min, respectively, as shown in Figure 2.
(a) LC chromatogram of (a) LNG (20 μg mL−1) and (b) EMG (40 μg mL−1) using UV detection at 226 nm; (b) LC chromatogram of (a) ALG (10 μg mL−1) and (b) PGN (9.7 μg mL−1, using UV detection at 270 nm
Citation: Acta Chromatographica Acta Chromatographica 29, 4; 10.1556/1326.2016.00089
For the second HPLC method, the simplest mobile phase was developed for this method using 50% methanol and adjusting to the pH to 2.7 using orthophosphoric acid. The flow rate was set to be 0.8 mL min−1 enhancing resolution between peaks by increasing the difference in their retention time. UV detection at the isosbestic point (270 nm) was the most sensitive wavelength for the determination of the two drugs simultaneously. Retention times for ALG and PGN were found to be 3.2 and 5.2 min, respectively, as shown in Figure 2.
3.2 System Suitability Tests.
System suitability tests are an integral part of liquid chromatographic methods in the course of optimizing the conditions of the proposed method. System suitability tests are used to verify that the reproducibility was adequate for the analysis performance. The parameters of these tests are number of theoretical plates (N), tailing of chromatographic peak (T), capacity factor (K), resolution between peaks (Rs), and repeatability as percent relative standard deviation (% RSD) of peak area and retention time for six injections as shown in Table 1.
System suitability tests for HPLC–UV methods for simultaneous determination of ALG and PGN and simultaneous determination of LNG and EMG
| Item | ALG | PGN | LNG | EMG | Limits |
|---|---|---|---|---|---|
| N | 1520 | 2809 | 3600 | 3580 | >2000 |
| T | 1.02 | 1.04 | 1.05 | 1.04 | ≤2 |
| k | 3.00 | 4.80 | 1.20 | 2.60 | ≥1 |
| Rs | 5.26 | 5.13 | >2 | ||
| % RSD of 6 injections Peak area Retention time | 0.16 0.23 | 0.29 014 | 0.30 0.26 | 0.15 0.18 | ≤2% ≤2% |
N indicates number of theoretical plates; T, tailing factor; K, capacity factor; Rs, resolution; RSD, relative standard deviation.
3.3 Methods' Validation According to ICH Guidelines [14]
3.3.1 Linearity.
A linear relationship between peak area and component concentration was obtained for six chosen concentrations of each drug, and the regression equations were computed. The analytical data of the calibration curves are summarized in Table 2. The validity of the calibration curves was confirmed by the obtained low values of LOD–LOQ parameters and acceptable values of STEYX, Sb, and Sa as shown in Table 2, where LOD is the limit of detection which represents drug concentration at STEYX/S ratio of 3.3, LOQ is the limit of quantification at which STEYX/S is 10, STEYX is the residual standard deviation of the regression line, Sb is the standard deviation of the slope, and Sa is the standard deviation of the intercept.
Results of assay validation of HPLC–UV methods for simultaneous determination of ALG and PGN and simultaneous determination of LNG and EMG
| Item | ALG | PGN | LNG | EMG |
|---|---|---|---|---|
| Range of linearity (μg mL−1) | 0.5–25 | 1–25 | 2–50 | 4–100 |
| Regression equation | Area × 10−5 = 0.8027C μg mL−1 + 0.4424 | Area × 10−5 = 0.751C μg mL−1 + 0.2048 | Area × 10−5 = 1.4329C μg mL−1 + 0.0213 | Area × 10−5 = 0.5738C μg mL−1 + 0.791 |
| Regression coefficient (r) | 0.9999 | 0.9999 | 0.9998 | 0.9999 |
| LOD (μg mL−1) | 0.15 | 0.22 | 0.49 | 0.79 |
| LOQ (μg mL−1) | 0.47 | 0.68 | 1.50 | 2.40 |
| aSb | 1.81 × 10−3 | 2.50 × 10−3 | 5.28 × 10−3 | 1.69 × 10−3 |
| aSa | 0.034 | 0.046 | 0.20 | 0.13 |
| Confidence limit of the slope | 0.8027 ± 0.027 | 0.7513 ± 0.035 | 1.4329 ± 0.28 | 0.5738 ± 0.072 |
| Confidence limit of the intercept | 0.4424 ± 8.03 × 10−4 | 0.2048 ± 5.11 × 10−4 | 0.0213 ± 1.12 × 10−4 | 0.791 ± 1.34 × 10−4 |
| Standard error of the estimation | 0.037 | 0.051 | 0.21 | 0.14 |
| Intraday precision % RSD | 0.12 – 0.16 – 0.29 | 0.11 – 0.25 – 0.37 | 0.16 – 0.27 – 0.32 | 0.17 – 0.19 – 0.26 |
| Interday precision % RSD | 0.15 – 0.21 – 0.33 | 0.19 – 0.22 – 0.35 | 0.13 – 0.24 – 0.28 | 0.15 – 0.22 – 0.30 |
| Robustness Flow rate (±0.1), % RSD | 0.23 | 0.19 | 0.20 | 0.11 |
| Organic strength (% ±2), % RSD | 0.17 | 0.38 | 0.31 | 0.18 |
| Mobile phase pH (±0.1), % RSD | 0.28 | 0.14 | NA | NA |
aSb indicates standard error of intercept; Sa, standard error of slope; NA: not applicable.
3.3.2 Accuracy.
Accuracy was calculated by % recovery of 5 different concentrations of each drug. The results including the mean of the recovery and standard deviation are shown in Table 3. In addition, accuracy was confirmed by % recovery of 9 different concentrations of each drug in the presence of the other drug in their laboratory-prepared mixtures. The results including the mean of the recovery and standard deviation are shown in Table 4. Furthermore, standard addition technique was applied to confirm and ensure the accuracy of the method and the concentrations were calculated using the corresponding regression equations as in Tables 5 and 6.
Results for simultaneous determination of ALG and PGN and simultaneous determination of LNG and EMG in bulk powder by the two proposed HPLC–UV methods
| ALG (μg mL−1) | aR % | PGN (μg mL−1) | aR % | LNG (μg mL−1) | aR % | EMG (μg mL−1) | aR % | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Pure | Found | Pure | Found | Pure | Found | Pure | Found | ||||
| 2.50 | 2.46 | 98.40 | 2.50 | 2.51 | 100.40 | 5.00 | 5.09 | 101.80 | 10.00 | 10.19 | 101.90 |
| 7.50 | 7.54 | 100.53 | 7.50 | 7.47 | 99.60 | 15.00 | 15.17 | 101.13 | 30.00 | 29.54 | 98.47 |
| 12.50 | 12.71 | 101.68 | 12.50 | 12.36 | 98.88 | 25.00 | 25.15 | 100.60 | 50.00 | 50.18 | 100.36 |
| 17.50 | 17.3 | 98.86 | 17.50 | 17.18 | 98.17 | 35.00 | 34.61 | 98.89 | 70.00 | 69.35 | 99.07 |
| 22.50 | 22.91 | 101.82 | 22.50 | 22.78 | 101.24 | 45.00 | 44.99 | 99.98 | 90.00 | 90.54 | 100.60 |
| Mean ± SD | 100.26 | Mean ± SD | 99.66 | Mean ± SD | 100.48 | Mean ± SD | 100.08 | ||||
| 1.58 | 1.21 | 1.11 | 1.35 | ||||||||
aMean of three determinations.
Simultaneous determination of ALG and PGN and simultaneous determination of LNG and EMG by two HPLC–UV methods in laboratory-prepared mixtures
| Ratio ALG–PGN | ALG (μg mL−1) | PGN (μg mL−1) | Ratio LNG–EMG | LNG (μg mL−1) | EMG (μg mL−1) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Pure | Found | aR % | Pure | Found | aR % | Pure | Found | aR % | Pure | Found | aR % | ||
| 5:1 | 25.00 | 25.50 | 102.00 | 5.00 | 4.91 | 98.20 | 5:1 | 50.00 | 50.09 | 100.18 | 10.00 | 10.09 | 100.90 |
| 4:1 | 20.00 | 19.69 | 98.45 | 5.00 | 5.09 | 101.80 | 4:1 | 40.00 | 39.98 | 99.95 | 10.00 | 9.92 | 99.20 |
| 3:1 | 15.00 | 14.80 | 98.67 | 5.00 | 4.97 | 99.40 | 3:1 | 30.00 | 29.87 | 99.57 | 10.00 | 10.17 | 101.70 |
| 2:1 | 10.00 | 10.06 | 100.60 | 5.00 | 5.05 | 101.00 | 2:1 | 20.00 | 19.77 | 98.85 | 10.00 | 10.15 | 101.50 |
| 1:1 | 5.00 | 5.05 | 101.00 | 5.00 | 5.07 | 101.40 | 1:1 | 10.00 | 10.20 | 102.00 | 10.00 | 9.97 | 99.70 |
| 1:2 | 5.00 | 5.07 | 101.40 | 10.00 | 9.89 | 98.90 | 1:2 | 10.00 | 10.17 | 101.70 | 20.00 | 20.38 | 101.90 |
| 1:3 | 5.00 | 5.08 | 101.60 | 15.00 | 15.07 | 100.47 | 1:3 | 10.00 | 10.06 | 100.60 | 30.00 | 29.54 | 98.47 |
| 1:4 | 5.00 | 5.03 | 100.60 | 20.00 | 20.38 | 101.90 | 1:4 | 10.00 | 9.98 | 99.80 | 40.00 | 40.20 | 100.50 |
| 1:5 | 5.00 | 4.94 | 98.80 | 25.00 | 25.06 | 100.24 | 1:5 | 10.00 | 10.12 | 101.20 | 50.00 | 49.92 | 99.84 |
| Mean ± SD | 100.35 | 100.37 | Mean ± SD | 100.43 | 100.41 | ||||||||
| 1.36 | 1.31 | 1.04 | 1.19 | ||||||||||
aMean of three determinations.
Simultaneous determination of LNG and EMG in pharmaceutical dosage form and standard addition technique by HPLC–UV method
| Pharmaceutical dosage form | HPLC method | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| a% Recovery ± SD | Standard addition technique | |||||||||
| LNG | EMG | Claimed concentration (μg mL−1) | Pure added (μg mL−1) | Pure found | a% R pure added | |||||
| LNG | EMG | LNG | EMG | LNG | EMG | LNG | EMG | |||
| Glyxambi® tablets (LNG and EMG) 5 mg/10 mg BN: 25061N | 99.91 ± 0.83 | 100.25 ± 0.92 | 20.00 | 40.00 | 10.00 | 20.00 | 9.88 | 19.65 | 98.80 | 98.25 |
| 15.00 | 30.00 | 14.89 | 29.42 | 99.27 | 98.07 | |||||
| 20.00 | 40.00 | 20.25 | 40.17 | 101.25 | 100.43 | |||||
| 25.00 | 50.00 | 25.49 | 50.77 | 101.96 | 101.54 | |||||
| 30.00 | 60.00 | 30.05 | 60.64 | 100.17 | 101.07 | |||||
| Mean ± SD | 100.29 ± 1.32 | 99.87 ± 1.61 | ||||||||
aMean of three determinations.
Simultaneous determination of ALG and PGN in pharmaceutical dosage form and standard addition technique by HPLC–UV method
| Pharmaceutical dosage form | a% Recovery ± SD | Standard addition technique | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| ALG | PGN | Claimed concentration (μg mL−1) | Pure added (μg mL−1) | Pure found | a% R pure added | |||||
| ALG | PGN | ALG | PGN | ALG | PGN | ALG | PGN | |||
| Oseni® tablets (ALG and PGN) 34 mg/33.06 mg BN: 25059N | 100.40 ± 1.51 | 100.31 ± 1.17 | 10.00 | 9.7.00 | 1.00 | 1.00 | 0.98 | 0.99 | 98.00 | 99.00 |
| 5.00 | 5.00 | 4.94 | 5.06 | 98.80 | 101.20 | |||||
| 10.00 | 10.00 | 9.92 | 10.08 | 99.20 | 100.80 | |||||
| 12.5 | 12.50 | 12.69 | 12.71 | 101.52 | 101.68 | |||||
| 15 | 15.00 | 15.30 | 14.81 | 102.00 | 98.73 | |||||
| Mean ± SD | 99.90 ± 1.76 | 100.28 ± 1.33 | ||||||||
aMean of three determinations.
3.3.3 Precision.
The % RSD values were calculated for the three concentrations of each drug mentioned under section Precision, within the same day and on three successive days, and found to be less than 1% as shown in Table 2.
3.3.4 Specificity.
Specificity was confirmed by % recovery of 9 different concentrations of each drug in the presence of the other drug in their laboratory-prepared mixtures. The results including the mean of the recovery and standard deviation are shown in Table 4.
3.3.5 Robustness.
There was no difference in the results obtained after small deliberate variations in some chromatographic conditions as change in the flow rate, the organic strength, and the value of pH of the mobile phase as shown in Table 2.
3.3.6 Pharmaceutical dosage forms and standard addition technique.
The proposed chromatographic methods were successfully applied to two pharmaceutical dosage forms. Standard addition technique was applied, and the concentrations were calculated using the corresponding regression equations as in Tables 5 and 6.
3.3.7 Limit of detection and limit of quantification for the proposed method.
Limit of detection (LOD) and limit of quantitation (LOQ) were determined for the proposed methods, and results are given in (Table 2).
3.4 Statistical Comparison.
Statistical comparison of the results obtained by the proposed methods and the reference methods [4 and 10] was carried out by “SPSS statistical package version 11” at P = 0.05 as shown in Table 7.
Statistical comparison between the proposed methods and the reference methods
| Statistical term | LNG | EMG | ALG | PGN | ||||
|---|---|---|---|---|---|---|---|---|
| Reference methodb | HPLC–UV method | Reference methodb | HPLC–UV method | Reference methodc | HPLC–UV method | Referencemethodc | HPLC–UV method | |
| Mean | 100.21 | 100.48 | 99.71 | 100.08 | 99.87 | 100.26 | 100.03 | 99.67 |
| ±SD | 1.29 | 1.11 | 0.97 | 1.35 | 1.30 | 1.58 | 1.54 | 1.21 |
| % RSD | 1.29 | 1.10 | 0.97 | 1.35 | 1.30 | 1.58 | 1.54 | 1.21 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| V | 1.66 | 1.23 | 0.94 | 1.82 | 1.69 | 2.50 | 2.37 | 1.46 |
| t (a2.306) | 0.36 | 0.50 | 0.43 | 0.41 | ||||
| F (a6.39) | 1.35 | 1.94 | 1.48 | 1.62 | ||||
aFigures in parentheses are the theoretical t values at (p = 0.05).
bReference method [6]: aliquots of standard solutions containing 10–50 μg mL−1 LNG and 1–32 μg mL−1 EMG were measured at 225 nm using LC–UV.
cReference method [12]: aliquots of standard solutions containing 5–25 μg mL−1 ALG and 10–50 μg mL−1 PGN were measured at 269 nm using LC–UV.
4. Conclusion
The proposed methods proved to be simple, accurate, and reproducible for the determination of LNG, EMG, ALG, and PGN, and they were validated showing satisfactory data for all the parameters tested. The developed methods can be conveniently used by quality control laboratories. The new methods were developed without use of any buffer applying simple mobile phases with the advantage of easy preparation and more sensitive results and offering mobile phases that are suitable for both UV detection and mass detection.
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