Newly developed method for the determination of sitagliptin phosphate using rubeanic acid as a precipitating agent via the use of ISNAG continues low luorimeter

A new, a simple, sensitive and fast developed method for the estimation of sitagliptin phosphate in pure and pharmaceutical drugs (tablet). This method based on the reaction between sitagliptin phosphate and rubeanic acid to form reddish-black ppt, using ISNAG-Fluorimeter analyzer via the measure of diverged light at 90° from radiation sourcewhich is used a range of high intensity ultraviolet 184.9 nm & 253.7 nm by low-pressure mercury lamp while detector cell can detect at 410-1150 nm. The resultant of diverged light by an interaction between incident photons and precipitated particles giving rise to a longer wavelength that the detector can detected. Chemical and physical parameters were studied and optimized. The calibration graph was linear in the range of (0.03-13 ) mmol.L−1 , with correlation coef icient ( r )= 0.9881 ,linearity percentage r%= 97.64 , L.O.D = 0.7848μg/sample and RSD lower than 0.6% for (3&7 ) mmol.L−1 ( eight replicates) concentration of sitagliptin phosphate. This method was successfully applied for the determination of sitagliptin phosphate in two different companies of pharmaceutical drugs. A comparisonwasmadebetween thenewlydevelopedmethod analysiswith the classical method (UV-spectrophotometer by measuring of absorbance ) using standard addition method via the use of t-test, it was noticed that there was no signi icant difference between two methods at %95 con idence level.


Molecular weight-523.32 gm/mol
It is the irst and simplest prescription medicinal drug in a novel magni icence of the oral antihyperglycemic agents, enhancing the ability of the body in lowering the glucose of the blood in the case where it is elevated. The therapeutic aggregate in the second Type is orally using lively inhibitors Dipeptidyl Peptidase-4 (DPP -IV) (Herman et al., 2007;Green et al., 2006) such as the Sitagliptin Phosphate. It's an oral anti-diabetic medication (Gallwitz, 2007;Aschner et al., 2006) that allow to govern the levels of the blood sugar through the regulation of the levels of insulin within the body. (Raz et al., 2006;Koenig et al., 1976).

Reagent and chemical
Every chemical has been used of the analyticalreagent and the distilled water turned into used for the preparation of every solution. A standard solution 0.1mol/L of sitagliptin phosphate molecular formula C 16 H 20 F 6 N 5 O 6 P, M.wt. 523.23 g/mole and SDI-Iraq become prepared through dissolving 5.2323 g in 100 ml of the distilled water. A stock solution 50 mmol/L of rubeanic acid molecular formula C 2 H 4 N 2 S 2 M.wt. 120.2 g/mole has been produced through the dissolving 3.005 g in 500 ml of sodium hydroxide (0.01N).

Sample preparation
20 tablets have been weight and later crushed and ground. Tablets which contain 100mg of sitagliptin phosphate have been weighted 2.8911 g, 2.17307g ( equivalent to 0.5232g of the active ingredient, 10 mmol/L) for sitavi-Iraq and Januvia-MSD-UK, respectively. Everyone from the two types of drug which have been dissolved in distilled water. This solution has been iltered in order to cast off the undissolved materials, the residue has been washed by the distilled water and the quantity has been completed to 100 ml by distilled water.

Apparatus
The response was measured by a homemade ISNAGluorimeter. A low-pressure mercury lamp is used, which is characterized by two lambdas (184.9 & 253.7) nm. While the detector that is been used a 2[4 x 2.5cm] solar cell. The low device used to determination of sitagliptin phosphate be seen inFigure 2. Peristaltic pump 2 channels with different speeds (Ismatec, Switzerland). Valve six-port medium pressure injection valve ( I D E X corporation, U.S.A. ) with the loop of the sample (1 mm i.d. Te lon, different length ).2[4 x 2.5cm] solar cells are utilized as a detector to collect the signal via sample travel through a line of 2mm optical aperture extended for 100mm distance. The signals of the output have been recorded through the potentiometric recorder (Siemens, Germany)( 1V-5 V, 1000 mV-5000 mV). The peak height has been evaluated for every one of the signals. UV-Spectrophotometer digital double beam type (UV-Vis spectrophotometer, UV-1800, Shimadzu, Japan) has been applied as well for scanning the sitagliptin phosphate spectrum, 4cm quartz.
A design system of manifold consisting of two lines ( Figure 2) was used.
The 1 st line is the carrier stream (KCl 0.07 mmol.L −1 , 1.5 ml/min) that will introduce the sample segment into the reaction stream by combined with the second line ( rubeanic acid 1.6 ml/min )to form a reddish-black ppt at Y-junction. The precipitate measured by using ISNAG-Fluorimeter via low-pressure mercury lamp it gives two main wavelengths, namely 184.9nm and 253.7nm. These both two lines are easily diverged due to its high frequency. The divergence of this beam of the incident light will be detected at 90 o through a low cell of 2mm path length that extends for 100mm distance by using 2[4 x 2.5cm] solar cell. Scheme 1 indicates a suggested mechanism for the reaction of sitagliptin phosphate -rubeanic acid.         A different concentration of rubeanic acid was used (0.1-3 mmol/L) as a precipitating agent at a low rate of 1.2 ml/min for each line, using 75 µL sample volume and water as a carrier stream. Figure 3-A shows the response pro ile and the result obtained in Table 1, it was observed that an increase of diverged light expressing of peak height up to 1mmol/L for rubeanic acid (Figure 3-A). More than 1mmol/L lead to form a small size of particles, which in turn to lead a decrease in the amount of diverged light. Therefore, 1mmol/L for rubeanic acid (Figure 3-B) is the most suitable concentration.

Reaction medium used as carrier stream
A different solutions media were study as a carrier stream (NaCl, NaNO 3 , KCl, KBr, NH 4 Cl& H 2 O ) in the reaction between STP (7 mmol/L) and rubeanic acid(1 mmol/L ) to form reddish-black ppt using sample volume 75 µL and low rate 1.2 ml/min for carrier stream & reagent. Figure 4-A, B shows that the best media is KCl compared with other salts as a carrier stream, it might be due to aggregation precipitating of particules and increase of size lead to increase of scattered light on the detector. But other salts as a carrier stream lead to a decrease of light might be due to the form of small-sized particulate because of uncompleted growth form of particles. The obtained results were summarized in Table 2.

Effectof Potassium chloride concentration
Using experimental parameters (7mmol.L −1 sitagliptin phosphate and 1mmol.L −1 rubeanic acid at 1.2 ml/min low rate of every line with 75 µL sample volume ).A variable concentration (0.05-0.5)mmol.L −1 of potassium chloride solution were prepared to obtain the prefect concentration of it as a carrier stream. It was noticed that 0.07 mmol.L −1 of KCl gives a height response; above that causes a decrease in the height of response( Figure 5-A,  B). This effect might be due to the dissociation of large particulate into small size than the falling wavelength, which leads to not detected by solar cells. The results were tabulated in Table 3.

Effect of low rate
A changble of low rates ranging (0.9-2)ml/min for a carrier line and ( 1.0-2.1) ml/min for reagent line were used for sitagliptin phosphate (7mmol/L,75µL )-rubeanic acid (1mmol.L −1 ) system and open valve mode. Figure 6-A shows at low low rate wide pro ile response were obtained, this might be attributed to the increased dispersion which in turn will increase the area of precipitated particle segment in a low cell . while at higher low rate; an increased peak response were obtained up to 1.3 ml/min followed by a decrease in response as not enough time is given to detect the variation in carrier stream composition. Figure 6-B shows that the low rate 1.5, 1.6 ml/min for carrier & reagent line will be used to obtain a regular response. The acquired results were summarized in Table 4.

Effect of sample loop & lag time
The study was carried out using variable sample volume ( Table 5.

Calibration graph
Using the optimum conditions, a set of STP concentrations vary from (0.03-15) mmol/L were prepared to require to prepare a scatter plot graph followed by choice of calibration graph, Figure 8-A shows response pro ile for this study. While Figure 7-B shows the variation of scattered diverged light using ISNAG-luorimeter with STP concentration. It was noticed that the linear calibration graph ranging from (0.03-13) mmol/L with correlation coef icient r: 0.9881 in which that an increase of STP concentration leads to an increase of solid crystalline precipitate having a smooth surface which works as a re lecting mirror to word the detector that will be measured at 90°according to instrument design. This method compared with the classical method through measurement of absorbance by UV-spectrophotometer at 267 nm (Anudeepa et al., 2015) , Figure 8-C. The results tabulated as Table 6. The repeatability was studied for the determination of STP via the measurement of diverged light at 90°f or the reaction of STP with rubeanic acid at the concentration (3 & 7 mmol.L −1 ), as shown in Figure 9.
The L.O.D of STP calculates through two methods. Gradual dilution of low concentration in the calibration graph or primarily based on the numerical value of slope as to be seen in Table 7.

The use of ISNAG -luorimeter for the determination of STP in the pharmaceutical preparation
The technique that has been adopted has been utilized to determine the STP in two different companies of pharmaceutical drugs (Sitavia, Januvia ). Continuous low injection analysis using homemade ISNAG-luorimeter using a mercury lamp tube of low-pressure (UV-Light) and detection of diverged scattered lights (visible light) At 2 X 90 o using multi solar cells that cover 2 X 100mm distance of 2mm path length. A set of solutions have been prepared for each one of the pharmaceutical drugs (0.01 mol/L).
Through the transfer of 1ml to every 5 volumetric lasks (10 ml ), followed with the aid of the addition of gradual standard STP volumes (0, 0.20, 0.30, 0.40, and 0.50) ml of 0.05mol/L for the sake of obtaining (0.0, 1.0, 1.50, 2.0 and 2.50 )mmol/L when use ISNAG-luorimeter, while transferring 1.25ml to every 5 volumetric lasks (25ml), followed with gradually adding standard STP volumes. (0,0.25, 0.35, 0.45 and 0.5) ml of 0.05mol/L for obtaining (0, 0.5, 0.7, 0.9 and 1) mmol/L for using UVspectrophotometer instrument (the conventional approach). The measures have been carried out with the two approaches. Figure 10-A&B&C has shown the response pro ile for this research and the calibration graphs of the standard addition with the use of the ISNAG-luorimeter. The results have been treated mathematically for the approach of the standard addition. Results have been listed in Table 8 at a 95% con idence level, which sho ws the practical concentration of the STP in every one of the pharmaceutical drugs with the use of 2 analysis approaches. Table 9 has illustrated a practical content of the active ingredient at a con idence level equal to95% &ef iciency of determination besides the paired t-test indicating a comparison at 2 different paths (Miller and Miller, 1988;Bluman, 1997).

First-The individual t-test
Comparing between a newly proposed approach with the use of ISNAG-Fluorimeter with a (100mg) of icial quoted value through the calculation of the t-value as has been listed in Table 9 column 6. It is worth to notice that calculating the t-value is less compared to the critical tabulated t-value, which implies the fact that there has not been any considerable difference between each individual company's quoted value with t cal at %95 con idence interval.
Second-The paired t-test has been utilized ef iciently for comparing between the developed approach the utilization of the ISNAC-Fluorimeter CFIA with the classical approach, the result that has been obtained, indicated that there has been simply no considerable difference between the 2 methods because the calculation of the t-value (-0.0732 ) has been less than t tab (4.303) for determining the STP in the pharmaceutical preparations at a con idence level of 95% as can be seen in Table 9.

CONCLUSIONS
The suggested method for the estimation of STP based on the formation of reddish-black for ionpair between the drug(STP) and rubeanic acid in KCl medium and measured the diverged light at 90°via the use of a homemade ISNAG-luorimeter. The technique is easy, sensitive, does not require reaction coil, costly chemical compounds and without involving any speci ic sample treatment. In addition to clean and inexpensive carryout. This technique used for determination of STP in nanograms for 150 µl sample volume in pure and pharmaceutical preparation.

ACKNOWLEDGEMENT
I would like to express my private gratitude to Prof. Dr. Issam M. Shakir Al-Hashimi for his considerable advice, precise comments, support and encouragement.

Con lict of Interest
None.

Funding Support
None.