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[An Official Publication of ISF College of Pharmacy, Moga]

Original Article
Year : 2018   |  Volume : 10   |  Issue : 3   |  Page : 113-120  

Effect of metformin and pioglitazone on serum albumin binding of selected sulfonylureas

Navjot Kaur Sandhu, Sukhbir Kaur, Durga Das Anghore, Pawan Kumar Porwal

Correspondence Address:Department of Quality Assurance, ISF College of Pharmacy, Moga, Punjab, India. Department of Pharmaceutical Chemistry, ISF College of Pharmacy, Moga, Punjab, India

Source of Support: Nil, Conflict of Interest: None declared

DOI: 10.4103/2231-4040.197331


Metformin (MET) and pioglitazone (PIO) are known to induce conformation changes in the tertiary structure of human serum albumin and inhibit the non-enzymatic glycation of albumin. The pharmacological doses of sulfonylureas are about 100–500 time less as compared to MET and about 10–20 time less than PIO. The liquid chromatography coupled with tandem mass spectrometry was used to quantify all analytes. The retention of all analytes was made on a C8 column (50 mm × 2.1 mm and 2.5 μm) using 10 mM ammonium acetate buffer in gradient elution with acetonitrile at 0.25 ml/min flow rate and quantitated at selective reaction monitoring mode for the respective analyte. The Amicon® ultrafiltration device was used to access albumin binding studies. The analytes were spiked to 4% recombinant human serum albumin (rHSA) and 10% glycated human serum albumin (Gly-HSA) corresponding to their peak plasma concentration. Finally, the rHSA and Gly-HSA were incubated with various levels of MET, PIO, and sulfonylurea/s. The optimized bioanalytical liquid chromatography–tandem mass spectrometry was found linear in the range of 5–2000 ng/mL for MET and PIO, whereas, the calibration curve for glibenclamide and glimepiride was made in the range of 5–500 ng/mL. The lower limit of quantitation (LLOQ) for glibenclamide and glimepiride was 0.25 ng/mL, and LLOQ values for MET and PIO were 0.50 ng/mL and 1.0 ng/mL, respectively, with sufficient accuracy and precision. Competitive inhibition of non-enzymatic glycation of rHSA was observed in the presence of MET and PIO. An alteration of protein binding capacity was observed for sulfonylurea when incubated with glucose plus MET plus and PIO in comparison to control.

Keywords: Albumin, liquid chromatography–tandem mass spectrometry, metformin, pioglitazone, protein binding, sulfonylurea

How to cite this article:
Sandhu NK, Kaur S, Anghore DD, Porwal PK. Effect of metformin and pioglitazone on serum albumin binding of selected sulfonylureas. Pharmaspire 2018;10(3):113-120.


Diabetes involves an alteration in several physiological conditions; thus, polypharmacy or multiple pharmacotherapies are prescribed to treat this pathophysiological effect. Patient prescribed with polypharmacy should be properly educated about their disease state and drug regimen prescribed them along with their family member.[1] Several drug-related and environmental factors such as Porwal1drug-drug interaction, fluctuation in drug plasma concentration error in prescribing the polypharmacy, probable/possible adverse drug effect, and enhanced multifold cost of medication are also affecting patient health/disease, when prescribed with multiple medications/prescriptions.[2-4] The first-line treatment for diabetes include biguanides with sulfonylurea/s, whereas third pharmacotherapeutic agents, namely glucagon-like peptide-1 analog/dipeptidyl peptidase-4 inhibitor, are often added as second-line therapy for aggressive control on diabetes.[5,6]
These multiple pharmacotherapies may induce a psychological effect on the diabetic patient. Duran et al. have considered concrete and valid instrument as stress in living with diabetes to those psychological effect in development and worsening disease status. Most of those pharmacotherapies are prescribed due to patient adherence.[7] Therefore, possibilities of “hypoglycemic shock” may increase several folds, especially in the treatment of the geriatric population with a sulfonylurea. These shocks may be continued for hours or even days and frequency may be 1 in a day to 1 in a week.[8]
Although alterations in protein binding have little or no clinical relevance[9] but increase in serum glucose level, induces conformational changes in serum albumin.[10,11] This conformational change in the serum albumin structure may be induced by physiological and pathological changes[12,13] as well as several chemical as well a therapeutic agents, namely metformin (MET),[14,15] sodium perfluorooctanoate,[16] tenoxicam,[17] tetracyclines,[18] and gemcitabine hydrochloride[19]. The conformational changes in the serum albumin structure may alter the drug plasma concentration, and the alteration in the pathophysiological state of glucose metabolism could precipitate impaired glucose intolerance (IGT).[20,21]
Therefore, it was thought worthy to observe the effect of MET and pioglitazone incubation on recombinant human serum albumin (rHSA) and its subsequent effect on protein binding capacity of sulfonylurea. The glycated albumin was also used to simulate the diabetic conditions. The ultra-high-performance liquid chromatography-tandem mass spectrometer (UHPLC-MS/MS) method was developed and validated for simultaneous estimation of MET, pioglitazone (PIO), glimepiride, and glibenclamide [Figure 1] as per USFDA guideline.[22]


Chemical and reagent

Qualified standards of glimepiride, glibenclamide, and PIO hydrochloride were a gift from Mcleod Laboratories Pvt. Ltd. (Mumbai, India). The qualified standards of MET hydrochloride, rHSA, and glycated human serum albumin (Gly-HSA) were purchased from Sigma-Aldrich (Bangalore, India). Analytical/HPLC grade chemicals and solvents were obtained from Ranbaxy Fine Chemicals Limited (Delhi, India). Amicon® Ultra centrifugal filtration devices (MWCO: 10 kDa) were purchased from Millipore (Bangalore, India).

Chromatographic conditions

The Dionex Ultimate 3000 RSLC UHPLC was coupled to Bruker maXis QTOF mass spectrometer. The HPLC is consisting of rapid separation module of multiple wavelength detector and diode array detector. The mass spectrometer was equipped with electrospray ionization and atmospheric pressure chemical ionization.
The simultaneous elution of MET, PIO, GLBN, and GLMR was achieved on an Acquity UPLC X-Bridge BEH C8 column (50 mm × 2.1 mm, 2.5 μm) using 10 mM ammonium acetate buffer in gradient elution with acetonitrile at 0.25 ml/min flow rate. The instrument was operated in positive electrospray (+ES) mode. Retention window was kept for 0.00 min to 5.00 min and dwell time was 0.50 s. The capillary voltage was 3.50 kV whereas, high voltage (HV) lens voltage was 0.8 V. Captopril and ranitidine were selected as internal standard (ISs), and detection of ions was accessed using selective reaction monitoring mode by monitoring the transition pair of precursor ion and production for MET, PIO, glibenclamide, and glimepiride at 130.1 > 70.9, 357.1 > 134.0, 494.3 > 369.3, and 491.3 > 352.5, respectively, using HyStar™ data integration software.
The HPLC-UV method development was performed on a HPLC system (JASCO, Kyoto, Japan) composed of a PU-2089 plus quaternary pump solvent delivery module, a manual rheodyne injector with a 20 μL fixed loop and a UV-2075 intelligent UV–visible detector. For UV-Spectroscopic measurements, the UV-spectrophotometer (Shimadzu UV-1800, Kyoto Japan) was used.
For statistical calculations in bioanalytical method validation GraphPad PRISM® version 5.1 for Windows (GraphPad Software Inc., California, USA) software were used.

Preparation of resolution solution

The stock solution of all the analytes having a concentration of 1000 μg/mL in a diluent (acetonitrile: water 50:50 v/v) was prepared separately. The prepared stock solution was stored at −20°C in an amber glass volumetric flask. The stock solution was appropriately diluted to get standard solutions within the required concentration range freshly before analysis. The resolution solution containing 10 μg/mL of each analyte as prepared from stock solution.

Sample processing

All in one solution containing all analytes (2 μg/mL of each analyte) was prepared from stock solutions. The blend was subjected to vortex for 2 min followed by addition of 75 μL of 10% trichloroacetic acid (TCA) solution and 355 μL of cold acetonitrile. The mixture was subjected to vortex for 30 s and centrifuged at 12,000 rpm for 5 min. About 250 μL of supernatant was collected and mixed with equal volume of mobile phase and subjected to vortex for 3 min. About 10 μL of the mixture was injected into liquid chromatograph.

Preparation of calibration curve solution

Calibration standards were prepared of concentrations 5, 10, 20, 50, 100, 250, and 500 ng/mL for GLBN and GLMR, whereas 5, 50, 100, 250, 500, 1000, and 2000 ng/mL for MET and PIO from a standard stock solutions by diluting with appropriate diluent. Calibration curves were then constructed by plotting the peak area against the concentration of analytes. The goodness of fit was accessed for each calibration curve, and weighing was applied, where applicable.

Incubation of rHSA with D (+) glucose, MET, and PIO

The glycated albumin was prepared as mentioned in literature[23] with in-house modification in procedure.[24] Incubated samples were compared to control samples which were prepared and incubated for same time period by dissolving identical quantities of albumin incubated with glucose in phosphate buffer and dialyzing against distilled water for 24 h. A 10% Glycated albumin was collected and used for further studies. To access the competitive binding of MET and PIO, the selected analytes at their specific plasma concentration range, () i.e., MET and PIO were spiked to glycated albumin (10%) to obtain the concentration of 2000 μg/mL and 1000 μg/mL, respectively. The samples were incubated for 7 days in capped, sterile vials at 37°C and 20 RPM in a metabolic shaker. The remainder was lyophilized and stored at −20°C until further usage.

Protein binding capacity of sulfonylurea

The protein binding studies were performed using ultrafiltration model. The LC-MS/MS method was used to quantify the concentration of GLBN and GLMR in in vitro rHSA samples for drug-protein binding study. Plasma protein binding studies of all analytes were performed using Amicon® Ultra centrifugal filtration devices (MWCO:10 kDa). GLBN and GLMR were spiked to rHSA solution, previously spiked with MET plus PIO, to achieve peak plasma concentration of each analyte. To achieve equilibrium between the drug and plasma proteins, the spiked protein samples were incubated at 37.4°C for at 20 rpm 90 min before ultrafiltration using Envirogeine® metabolic shaker. Samples of 0.5 mL volume were transferred and centrifuged at 2500 g for 20 min at 4°C. Approximately, 200 μL of the ultrafiltrates were then collected. The ultrafiltrate collected in receiver compartment was injected to the LC-MS/MS and the non-specific binding was calculated using following Equation 1.



LC-MS/MS method development

Initially, the HPLC-UV method was developed for simultaneous estimation of MET, PIO, GLBN, and GLMR. The physicochemical properties of MET, PIO, GLBN, and GLMR are varied; therefore, simultaneous elution of selected analytes within stipulated/short retention time was also difficult in HPLC-UV method as shown in Figure 2. In addition, as per the EMEA guideline, the sensitivity of an analytical method should be able to quantify at 5% of peak plasma concentration with good accuracy and precision for routine sample analysis.[25] Therefore, mass spectrometric method was employed to quantified selected analytes for better sensitivity. During mass spectrometric method development, the electrospray ionization parameters were tuned in both negative and positive ionization mode for the selected analytes using as tuning solution 10.0 ng/mL of every analyte. Optimal responses were obtained for analytes in positive ionization mode as the selected actives are basic in nature due the presence of secondary and tertiary amino groups in chemical structure. The analytes and IS gave predominant singly charged protonated precursor ion [M+H]+ at m/z at 130.1, 357.1, 494.3, 491.3, and 217.2 for MET, PIO, glibenclamide, glimepiride, and captopril, respectively, in the first full scan. Further, fragmentation of the precursor ion was instigated using sufficient nitrogen for collisionally activated dissociation and by applying 32.0 psi curtain gas to break the precursor ion. The most copious and reproducible daughter ion in resultant mass spectra of actives were found at 70.9, 134.0, 369.3, 352.5, and 115.9 for MET, PIO, glibenclamide, glimepiride, and captopril, respectively. The mass spectra of selected analytes were shown in Figure 3. The nebulizer gas pressure was set at 55 psi to obtain ideal Taylor cone for a better spectral response. Capillary voltage and HV lens voltage did not have any significant effect on analytes response; therefore, these were maintained at 3.5kV and 0.8kV, respectively. The dwell time of 500 ms was found sufficient for the collection of analytes response. The column is operated at room temperature (22–25°C). A split of the column eluant of 1:10 was included so that only approximately 25 Μl/min entered the mass spectrometer.
The chromatographic elutions were set to achieve adequate retention and resolution of analytes from matrix disturbances. The mobile phase, for the chromatographic method, was optimized in the terms of composition, flow rate, and buffer strength. Different types of acidic buffers, namely ammonium acetate/acetic acid, ammonium formate/formic acid of different strength (2.0, 5.0, and 10.0 mM) in combination with acetonitrile/methanol were tested as the mobile phase. Further, the mobile phase modifier/additives such as ammonium formate, formic acid, and ammonium trifluoroacetate were also attempted to improve peak shape and retention on a C18 stationary phase. Several reversed phase stationary phases with the difference in carbon loading, porosity, and dimensions were tried with a flow rate in the range of 0.2–0.8 mL/min, which was responsible for satisfactory chromatographic peak shape and retention. The elution of MET was too early whereas, the peak shape was improper (i.e., high in tailing) for highly lipophilic drugs, namely PIO, glibenclamide, and glimepiride. The C8 columns provided acceptable retention and resolution of analytes within 3.5 min. Moderately non-polar stationary phase was selected for elution of selected analytes, whereas, organic content in mobile phase was kept high (~60%) for shorter elution time of selected analytes. Therefore, a mixture of acetonitrile: methanol (90:10, v/v) and 2.0 mM ammonium formate in the ratio of 95:5 were eluted through a C8 column (50 mm × 2.1 mm and 1.7 μm) at a flow rate of 0.5 mL/min. The peak shape and retention were consistent and percentage RSD of peak area and retention time was below 2.0. In addition, the responses were consistency at lower limit of quantitation (LLOQ), low quality control (LQC), middle quality control (MQC), and high quality control (HQC) levels for all analytes. The retention time for MET, PIO, GLBN, and GLMR was 1.11, 2.79, 3.04, and 3.14 min, respectively, as shown in Figure 4. The retention of ranitidine and captopril was observed at 1.68 and 2.23 min, respectively, whereas, the mother ion and daughter ions for ranitidine and captopril (ISs) were detected at 315.02 > 176.15 and 217.20 > 115.90, respectively.
Based on similar extraction efficacy and elution pattern, captopril, angiotensin-converting enzyme inhibitor was selected as IS for lipophilic compounds, i.e., PIO, glimepiride, and glibenclamide, whereas ranitidine, having log P < 2.0, was taken as IS to MET. Both ISs did not affect analytes sensitivity, recovery, and ion suppression.
The protein precipitation was the favored choice for extraction of drugs from biological fluids because of the minimized steps in extraction and less cost involved in the extraction process. The method was attempted using cold aqueous solution of 10% TCA and aconitase (ACN) and combination thereof. Human plasma (200 μl) was spiked with all-in-one working stock solution of all analytes, with working stock solution of ISs to get a concentration of 10 ng/ml of all actives and IS. MET has shown maximum recovery when TCA was used as precipitating agent where the recovery values for lipophilic analytes were <50%. We had observed opposite pattern when acetonitrile was used as precipitating media. Therefore, in the precipitation media were developed to obtain highest recovery of all analytes using combination of TCA and ACN. The precipitation was carried out using 1 part of plasma, 0.1 part drug and 0.05 parts of ISs. Diluted standard solution containing all analytes (15 μL) was added to 150 μL of plasma previously spiked with ISs in a 1.5 mL capacity microcentrifuge tube. The blend was subjected to vortex for 5 min. The blend was allowed to cool, and about 335 μL of ACN was added followed by centrifugation for 10 min at 15,000 rpm at 4°C. The blend was stabilized and allowed to cool for 10 min followed by complete dryness under nitrogen. The sample was reconstituted with an equal volume of mobile phase and subjected to vortex for 2 min. The supernatant was collected and injected into chromatograph to both HPLC-UV [Figure 5] and LC-MS/MS system. The percentage mean recovery for glimepiride and glibenclamide was 88.5 and 90.1%, respectively.

Specificity and sensitivity

To access specificity, the plasma collected from two different humans (untreated with any drug since last one month) was extracted with and without IS. The glimepiride and glibenclamide and IS analytes were well separated from the interferences under the optimized chromatographic conditions. The peaks were of good in shape, completely resolved from plasma components. Selectivity was evaluated by comparing the mean peak response at the Rt of glimepiride and glibenclamide obtained in blank plasma samples to that of mean peak response of the glimepiride and glibenclamide at LLOQ. The LLOQ for glibenclamide and glimepiride was 0.25 ng/mL, and LLOQ values for MET and PIO were 0.50 ng/mL and 1.0 ng/mL, respectively, with sufficient accuracy and precision. The mean response for the peak in two blank human plasma samples at the Rt of the glimepiride and glibenclamide was <20% of mean area for the glimepiride and glibenclamide peak at the assay sensitivity limit of 20 and 50 ng/mL, respectively. The mean response for the peak in two blank human plasma samples at the Rt of the all analytes was <5% of mean area for the valsartan peak at the IS level, 5 μg/ml. It has indicated that proposed method was highly selective and concentrations down to the LLOQ were detected with acceptable accuracy and precision using this method (CV% and RE% < 15%). The mean background response of blank was also calculated and found to be <20% of the response at the limit of quantification.

Calibration curve

The standard curve was determined on each day of the 6 days validation period; the slope, intercept, and the correlation coefficient were determined. Each run consisted of a system suitability sample, blank samples (a plasma sample processed without an IS), a zero sample (a plasma processed with IS), calibration curve consisting of 6–8 non-zero samples covering the total range (5–500 ng/mL for glimepiride and glibenclamide), and quality control (QC) samples at three concentrations (n = 6, at each concentration). Such runs were generated on 6 consecutive days. Calibration samples were analyzed from low to high at the beginning of each run, and other samples were distributed randomly through the run. The calibration curves plotted between concentration and peak area ratio, and goodness of fit was observed. The mean relative error (n = 6) was calculated at LLOQ level for both analytes. The extraction recovery was calculated at LLOQ, QC samples, upper limit of quantitation level (n = 6). The highest recovery at 10 μg/mL concentration was obtained for glimepiride and glibenclamide (~90.0%) using protein precipitation method when cold acetonitrile was used. The recoveries of glimepiride were ranged from 89.52% to 95.88% in ACN whereas the recoveries of glibenclamide were ranging from 90.11% to 95.39%.

Precision and accuracy

Calibration standards were prepared of concentrations 5, 10, 20, 50, 100, 250, and 500ng mL-1 for GLBN and GLMR whereas 5, 50, 100, 250, 500, 1000, and 2000 ng/mL for MET and PIO from a standard stock solutions by diluting with appropriate diluents.
Intraday precision, interday precision, and the accuracy were calculated from data obtained during a 6-day validation. Three concentrations were chosen from the high, medium, and low range of the standard curve as QC samples. Plasma samples spiked at five concentrations, i.e., LQC, MQC, and HQC were analyzed at each day of the 6-day validation (n = 6 at each concentration). The concentration representing LQC, MQC, and HQC level was 25, 100, and 400 ng/mL, respectively, for selected sulfonylureas. The concentration representing LQC, MQC, and HQC level was 25, 500, and 1000 ng/mL, respectively, for MET and PIO.


Stock solutions of all analytes and their ISs were stable at room temperature for 24 h and at 2–8°C for 48 h. The analytes in control human plasma at room temperature were found stable at least for 24 h and for minimum of three freeze and thaw cycles. Spiked plasma samples, stored at −20°C for long-term stability experiment, were stable for minimum of 90 days. The results of bioanalytical method validation for glimepiride and glibenclamide were summarized in Table 1.

In vitro protein binding studies for glibenclamide and glimepiride in the presence of MET and PIO

Although MET does not bound to human serum albumin (albumin binding ≈ 4–5%), it inhibits non-enzyamtic glycation process[26] and induces conformation changes in the human serum albumin’s structure.[14] PIO binds to serum albumin to a greater extent (>95%) and a powerful inhibitor of advanced glycation end product (AGE) formation,[27-29] but the mechanism of inhibition is still unknown. The members of thiazolidinediones family produce conformational changes in peroxisomal proliferator-activated receptor-γ, and one of the probable mechanisms may involve direct interaction between hydrazine nitrogen groups in carbonyl group.[30]. MET inhibits glycation of serum albumin protein and known to induce conformational changes in the albumin structure and trap the circulating methylglyoxal and/or other dicarbonyl compounds to reduce the formation AGEs.
We had observed an alteration in the serum albumin binding pattern of sulfonylurea in the presence of MET and PIO. The percentage protein binding of selected sulfonylureas has been detailed in Table 2.
It has been observed that the free plasma concentration of selected sulfonylureas has been increased to 10–15% as compared to control samples not spiked with MET and PIO. The alteration in free concentration of may fluctuate the blood glucose level which ultimately results in precipitation of IGT in diabetic patient especially on multiple therapies. Therefore, dose of glimepiride and glibenclamide should be precisely decided and adjusted when prescribed in combination with MET and PIO.




Glycation and MET induce conformational change in serum albumin structure, but the mechanism of coeffectiveness of MET, PIO, and sulfonylurea combination is still unknown. Alteration or regular fluctuation of serum concentration of sulfonylurea may result in IGT. Thus, a simple, fast, accurate, and precise bioanalytical LC-MS/ MS method was developed and validated to quantify small amount of MET, PIO, glimepiride, and glibenclamide in ultrafiltrate. The validated LC-MS/MS was able to qualify all analytes at sub-nanogram level with sufficient accuracy and precision. The extraction recovery for all the analytes was in the range of 80–90%. The validated bioanalytical method was found linear, precise, and accurate in the said range. Thus, it can be concluded that binding of MET and pioglitazone had affected serum albumin binding of glimepiride and glibenclamide in similar fashion which was independent to the stage of glycation.


The authors are thankful to BCUD (University of Pune, Grant no.: 13PH000692) for funding this research work. We are also thankful to Mylan Pharmaceutical Pvt. Limited (Nasik, India) for providing gift samples of active pharmaceutical ingredients.

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