Visible Spectrophotometric Determination of Gemigliptin Using Charge Transfer Complex

A visible spectrophotometric method was developed and validated for the determination of gemigliptin present in bulk drug and tablet formulation. It involves an indirect method of charge transfer complex formation in presence of NBS, metol and suphanilic acid. Gemigliptin was subjected to oxidation with excess amount of oxidant (NBS) and the unconsumed NBS oxidizes metol to give p-N-methylbenzoquinone monoamine (PNMM) which in turn forms a charge transfer complex with sulphanilic acid. Then validated the above developed method as per the current ICH guidelines. An excellent correlation coefficient (> 0.999) was found for the obtained regression equation (y = –0.0302x + 0.928) in the range of 2.0–30.0 μg mL-1. The method was found to be simple and rapid because it does not involve any solvent extraction. The recovery levels of the drug were in the range 99.92 – 100.08. keywords: Gemigliptin, Validation, Determination, Metol, Charge transfer complex.


INTRODUCTION
Gemigliptin is a selective, competitive and potent anti-hyperglycemic agent which is useful in the treatment of type 2 diabetes.It belongs to DPP-4 (dipeptidyl peptidase-4) inhibitor class 1 .Secretion of glucagon is decreased by it.Either as a monotherapeutic agent or in combination with metformin, it is effective 2 .It is administered orally.Literature survey reveals that HPLC-Isocratic [4][5] and LC/MS-MS 6 methods were developed for the assay of gemigliptin.However, visible spectrophotometric method was not proposed.Hence, a visible spectrophotometric method is developed and validated in the present study for the determination of gemigliptin with the help of a chromogen like metol.a working solution of 1 mg mL -1 .The solution was stored in a refrigerator.

Recommended analytical procedure
The important step in the present study is the fixation of upper concentration limit of oxidant.It was established by the treatment of various concentrations of oxidant with metol as well as sulphanilic acid.Into a sequence of 10 mL volumetric flasks, transferred different aliquots (0.2 -3.0 mL) of standard gemigliptin solution (100 μg mL -1 ) with the help of a micro burette.Then diluted to 3 mL by the addition of water.Then added one mL each of buffer (pH 1.5) and NBS (1 mg mL -1 ) using micro burettes.Mixed the contents well and kept aside for about ten minutes with occasional shaking.In a sequence, added one mL each of metol (0.2%) and sulphanilic acid (0.2%) with a time gap of one minute.Made up to the mark with water and shaken well.Measured the absorbance against blank at 631 nm after 10 min but before 90 minutes.

Chemicals and Instrumentation
A n a l y t i c a l g r a d e c h e m i c a l s w e r e used throughout the study and solutions were prepared using distilled water.A double beam spectrophotometer (Shimadzu UV-1700) was used along with Shimadzu UV-Probe 2.10 software.Standard quartz cuvettes were used for analysis.

Chromophore Formation and Chemistry
Out of the various reactions involved in spectrophotometric assay of pharmaceutical drugs, charge transfer reaction is proved to be highly sensitive as well as acceptable method, in addition to reactions involving to ion pair [9][10][11][12][13][14][15] and oxidation [16][17][18][19][20] .The complex formed between electron donor and acceptor compound yields a new absorption band with a precise l max pertaining to that complex.The reaction between oxidized form of metol and pharmaceutical drugs forms n-π* charge transfer complex 21 .In general the oxidized form of metol (N-methyl-p-aminophenol sulfate) acts as π-acceptor and forms a chromophore with primary aromatic amine containing drugs (n-donor) through a charge transfer reaction [21][22][23][24] .According to Amin et al., 25 , a ternary complex is formed between oxidant, metol and primary aromatic amine.The

Preparation of standard gemigliptin solution
The standard drug of gemigliptin (50 mg) was weighed accurately and transferred to 50 mL volumetric flask.It was dissolved properly and diluted up to the mark with methanol to obtain a final concentration of 1000 μg mL -1 (stock solution).This solution was further diluted suitably.

Buffer (pH 1.5)
Equal volumes of hydrochloric acid (one normal) and sodium acetate (one normal) were mixed and pH was adjusted to 1.5 with variation of either of them 7 .

N-bromosuccinimide (1 mg mL -1 )
About 0.5 g of NBS was dissolved in hot water.After filtration the solution was made up to mark in 250 mL volumetric flask to get ~ 0.01M NBS solution.Then standardised the solution by iodometry 8 .It was diluted appropriately to obtain amine involved in ternary complex felicitates the transfer of electrons between oxidant and metol.In acidic medium, the role of primary aromatic amine is evident from the accomplishment of reaction in its presence only 24 .Literature survey shows that many drugs bearing nitro group were reduced to concerned amines and then allowed to form a charge-transfer complex with the oxidised form of metol 21,[26][27][28][29] .Similarly, Hydrolysis of amide group resulted in to corresponding amines and then allowed to react with oxidized form of metol 29 .
Formation of charge transfer complex between oxidized form of metol and drugs containing aromatic amine group can be adopted when the drug is not oxidizable 25 .However, gemigliptin is easily oxidizable.Hence, an indirect method is adopted.In this method, a known amount of excess oxidant is added to gemigliptin in buffer medium where the drug is subjected to oxidation.The amount of unconsumed oxidant is determined by allowing it to interact with metol and sulphanilic acid, a primary aromatic amine.By the addition of gemigliptin in the order of increasing concentration to a fixed amount of oxidant, consumption of oxidant and hence, results in a consequent reduction in concentration of oxidant.Therefore, an increase of gemigliptin concentration causes a relative decrease in absorbance values.The mechanism involved in the present case corroborates with that of previous researchers 26,28,[30][31] .The plausible mechanism is shown in Fig. 2, which involves the in situ generation of p-N-methylbenzoquinone monoamine (PNMM) by the oxidation of metol.PNMM acts as an excellent electron acceptor and participates in the formation of a charge transfer complex with an electron donor like aromatic amines.
Simultaneous overlapping of orbitals of two PNMM molecules with a primary amine molecule is facilitated by the possible hydrogen which brings them closer.Formation of a charge transfer complex can be attributed to the transfer of electrons from HOMO (π) of aromatic primary amine to the LUMO (π*) of PNMM, the oxidized form of metol [32][33] .
The coloured charge transfer complex shows l max at 631 nm and the absorption spectrum is shown in Figure 3.

Validation of Method Linearity and range
An increase of gemigliptin concentration causes a decrease in concentration of unconsumed NBS leading a decrease in amount of formed PNMM.Hence, a decrease in absorbance was observed with an increase in gemigliptin concentration to give a linear curve with negative slope in the concentration range of 2.0-30.0μg mL -1 (Table 1, Fig. 4).The obtained linear regression equation y= -0.0302x + 0.928 possess a high correlation coefficient (> 0.999), indicating the linearity of the proposed method for determination of gemigliptin.Different parameters (optical and regression) are listed out in Table 2.

Accuracy
Accuracy of the current method was confirmed by determining %recovery values.In order to study various recovery levels at 50%, 100% and 150%, different amounts of gemigliptin (4.0, 8.0 and 12.0 μg mL -1 ) respectively were added to a nominal amount of (8.0 μg mL -1 ).Table 3 shows that 99.92 -100.08 is range of % recovery values.Low values for SD and %RSD reveal the acceptable level of accuracy.

Precision
Precision studies (intraday as well as inter-day) were studied by selecting three diverse gemigliptin concentrations in the linearity range (2.0-30.0μg mL -1 ).Table 4 shows the compilation of the six values measured each on the same day as well as successive days.Since the %RSD values are within tolerable limit (below 1%), the current method is validated in terms of both for intraday and inter-day precision studies.

Ruggedness
T hree dissimilar gemigliptin concentrations were chosen with in the linearity range (2.0-30.0μg mL -1 ) by two separate analysts on different days to carry out the experiments.The reproducible assay values are the evidence for ruggedness of the proposed method (Table 5).Apparent molar absorptivity (l mol -1 cm -1 ) 1.5 × 10 4 2.

Detection limits determination
Signal to noise ratio values were used to calculate both the limits for quantification as well as detection 30 .LOQ as well as LOD for gemigliptin determination using the current method was done using values of S (calibration curve slope) and σ (response standard deviation) 31 .LOD = 3.3 × σ /S = 0.010 μg mL -1 and LOQ = 10 × σ /S = 0.033 μg mL -1

Pharmaceutical Formulations Analysis
API present in tablet formulation (ZEMIGLO®) was extracted by sonnicating for a period of 10 min in presence of methanol.The above developed method was adopted to determine the API amount (Table 6).As visible spectrophotometry is preferred one in QC laboratories of developing countries [36][37] , current method can be implemented for routine assay of gemigliptin in pure and tablet formulations.

CONCLUSION
An indirect method of charge transfer complexation was successfully applied for the determination of gemigliptin.The proposed method was validated as per the existing guidelines of ICH.Being a substitute to the other expensive and sophisticated instrumental methods involving HPLC /LCMS-MS, current method can be adopted for routine assay of gemigliptin (bulk drug and tablet formulation) in quality control laboratories.

Table 4 : Precision data
* Average of six determinations

Table 6 : Assay of Pharmaceutical Formulation
* Average of three determinations