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Spectrochimica Acta Part A 68 (2007) 516–522

Simple, sensitive, selective and validated spectrophotometric methodsfor the estimation of a biomarker trigonelline from polyherbal gels

Shruti Chopra ∗, Sanjay K. Motwani, Farhan J. Ahmad, Roop K. KharDepartment of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India

Received 1 October 2006; received in revised form 11 December 2006; accepted 16 December 2006

bstract

Simple, accurate, reproducible, selective, sensitive and cost effective UV-spectrophotometric methods were developed and validated for the esti-ation of trigonelline in bulk and pharmaceutical formulations. Trigonelline was estimated at 265 nm in deionised water and at 264 nm in phosphate

uffer (pH 4.5). Beer’s law was obeyed in the concentration ranges of 1–20 �g mL−1 (r2 = 0.9999) in deionised water and 1–24 �g mL−1 (r2 = 0.9999)n the phosphate buffer medium. The apparent molar absorptivity and Sandell’s sensitivity coefficient were found to be 4.04 × 103 L mol−1 cm−1

nd 0.0422 �g cm−2/0.001A in deionised water; and 3.05 × 103 L mol−1 cm−1 and 0.0567 �g cm−2/0.001A in phosphate buffer media, respectively.hese methods were tested and validated for various parameters according to ICH guidelines. The detection and quantitation limits were found toe 0.12 and 0.37 �g mL−1 in deionised water and 0.13 and 0.40 �g mL−1 in phosphate buffer medium, respectively. The proposed methods were

uccessfully applied for the determination of trigonelline in pharmaceutical formulations (vaginal tablets and bioadhesive vaginal gels). The resultsemonstrated that the procedure is accurate, precise, specific and reproducible (percent relative standard deviation <2%), while being simple andess time consuming and hence can be suitably applied for the estimation of trigonelline in different dosage forms and dissolution studies. 2006 Elsevier B.V. All rights reserved.

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eywords: Trigonelline; Spectrophotometry; Method validation; Pharmaceutic

. Introduction

Chromatographic techniques like high performance thin-ayer chromatography (HPTLC) and high performance liquidhromatography (HPLC) have been extensively used to generateprofile of various chemical constituents of herbal drugs, i.e.

hemoprofiling. Specific organic solvents are used to isolatend identify the chemical constituents of herbs specific tohat particular species (e.g. alkaloid, glycoside, etc.) in anncreasing order of polarity and is characterized as a chemicalarker. When these chemical marker compounds possesses

n intrinsic biological activity of their own, they are knowns biomarkers. Biomarkers have gained increased attentionrimarily in European nations and in countries like China and

ndia, which have a long history of using herbal medicines andhe European Scientific Cooperative on Phytotherapy (ESCOP)as clearly specified the requirement of the standardization of

∗ Corresponding author at: Department of Pharmaceutics, Faculty of Phar-acy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India. Tel.: +91 11

605 9688; fax: +91 11 2605 9663.E-mail address: shrutichopra21@yahoo.com (S. Chopra).

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386-1425/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2006.12.021

mulations; Polyherbal gels

hytopharmaceuticals on the basis of biomarkers that arenique to that species [1].

Chemically, trigonelline is 1-methylnicotinic acid (Fig. 1)nd is an active alkaloidal constituent of Trigonella foenum-raecum, an aromatic herbaceous plant widely known forts various pharmacological activities [2–6]. Traditional valuef trigonelline can be attributed to its variety of medicinalroperties including antiseptic, antimigraine, antitumor andutagenic properties [7,8] and hence it can be used as a

iomarker for Trigonella foenum-graecum.Polymers have been widely used in drug delivery systems,

here they either act as a carrier for the drug or providehe desired attributes of the dosage form such as sustainedelease, prolonged release or mucoadhesive dosage forms, etc.evelopment of a suitable analytical method for determinationf trigonelline in presence of other herbal drugs and excipientss highly desirable.

Literature survey reveals the application of spectropho-

ometric [9] and high performance liquid chromatographyHPLC) method for the determination of trigonelline inrigonella foenum-graecum and in biological fluids [10].smotic pressure liquid chromatography (OPLC) [11,12],

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igh performance thin layer chromatography (HPTLC) [13]nd thin layer chromatography (TLC) are the other analyticalethods, which find application in trigonelline analysis.owever, chromatographic techniques are time consuming,

ostly and require expertise. Development of simple andccurate UV-spectrophotometric methods can provide a veryseful alternative for routine analysis of bulk, formulations andissolution samples.

The present research aimed at developing simple, accu-ate, sensitive, more convenient, less time-consuming andconomical UV-spectrophotometric methods for routine anal-sis of the trigonelline in pure form, their pharmaceuticalormulations and in vitro dissolution studies of vaginal gels andablet formulations. Two analytical methods have been devel-ped in different media for estimation of trigonelline. Mediased were deionised water and phosphate buffer (pH 4.5).rigonelline showed absorption maxima at 265 nm in deionisedater and at 264 nm in phosphate buffer (pH 4.5). The developed

nalytical methods were validated as per ICH guidelines andSP requirements [14,15]. Suitable statistical tests were appliedn validation data [16,17]. The present paper thus describes fast,imple, sensitive and validated UV-spectrophotometric methodor the determination of trigonelline.

. Experimental procedures

.1. Material and reagents

Standard trigonelline hydrochloride was purchased fromigma Aldrich Chemicals Pvt. Ltd., New Delhi, India.rigonella foenum-graecum was procured from the Green Earthroducts (New Delhi, India) and was assessed biologically by

he Department of Botany, Jamia Hamdard. Gum based- andolymer-based bioadhesive vaginal gels and vaginal tabletsere prepared in the laboratory. Apart from common excipients,

ablets contain excipients like carbopols (934P, 971P and 974P)kindly provided as gift samples by Noveon Inc., Mumbai,ndia), hydroxypropylmethyl cellulose (HPMC) and sodiumarboxymethyl cellulose. Vaginal gels contain excipients likearbopols, honey, polyethylene glycol (PEG), xanthan gumnd glycerin. All other chemicals and reagents used were ofnalytical grade and were purchased from Merck ChemicalsMumbai, India).

.2. Instruments

A double-beam Shimadzu 1601 UV–vis spectrophotometerShimadzu Corporation, Tokyo, Japan), connected to computer

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ta Part A 68 (2007) 516–522 517

nd loaded with UV-Probe software was used. For inter-ediate precision study, a different Shimadzu 1601 UV–vis

pectrophotometer connected to computer with UV-PC softwareas used. Both the instruments have an automatic wavelength

ccuracy of 0.1 nm and matched quartz cells of 10 mm (1.0 cm)ell path length.

.3. Analytical method development

Different media were investigated to develop a suitable UV-pectrophotometric method for the analysis of trigonelline inormulations. For selection of media, the criteria employed wereensitivity of the method, ease of sample preparation, solubilityf the drug, cost of solvents, applicability and robustness of theethod to various purposes. Absorbance of trigonelline in the

elected medium at respective wavelength was determined andpparent molar absorptivity and Sandell’s sensitivity coefficientsere calculated according to the standard formulae.

.4. Procedure for calibration curve

Two different stock solutions of 100 �g mL−1 of trigonellineere prepared in deionised water and phosphate buffer (pH.5) by dissolving 5 mg of trigonelline in 50 mL of each media.or preparation of different concentrations, aliquots of stockolutions were transferred into a series of 10 mL standardolumetric flasks and volumes were made with the respectiveedia. Six different concentrations were prepared in the range

f 1–20 �g mL−1 of trigonelline in the deionised water fortandard curve. In a similar way, six different concentrationsere prepared in the range of 1–24 �g mL−1 of trigonelline inhosphate buffer. Trigonelline was estimated at 265 and 264 nmn deionised water and phosphate buffer medium, respectively.

.5. Analytical method validation

.5.1. Specificity and selectivityTrigonelline solutions (10 �g mL−1) were prepared in

oth the selected media along with and without commonxcipients (lactose, microcrystalline cellulose, magnesiumtearate, talc, HPMC, carbopols (934P, 971P and 974P), sodiumarboxymethyl cellulose, honey, glycerine, PEG-400 andanthan gum, separately. All the solutions were scanned from50 to 200 nm at a speed of 400 nm min−1 and checked forny change in the absorbance at respective wavelengths. In aeparate study, drug concentration of 10 �g mL−1 was preparedndependently from pure drug stock in selected media andnalyzed (n = 9). Paired t-test at 95% level of significance waserformed to compare the means of absorbance (Table 1).

.5.2. AccuracyTo determine the accuracy of the proposed methods, differ-

nt levels of drug concentrations—lower concentration (LC),

ntermediate concentration (IC) and higher concentration (HC)in both media) were prepared from independent stock solutionsnd analyzed (n = 9). Accuracy was assessed as the percentageelative error and mean percentage recovery (Table 2). To

518 S. Chopra et al. / Spectrochimica Acta Part A 68 (2007) 516–522

Table 1Optical characteristics, statistical data of the regression equations and validation parameters for trigonelline (n = 9)

Parameter Deionised water Phosphate buffer, pH 4.5

Optical characteristicsApparent molar absorptivity (L mol−1 cm−1) 4.04 × 103 3.05 × 103

Sandell’s sensitivity (�g cm−2/0.001A) 0.0422 0.0567

Regression analysisSlope (S.E.a) 0.0233 (3.24 × 10−5) 0.0176 (3.07 × 10−5)95% Confidence limits of slope 0.02320; 0.02335 0.01750; 0.01765Intercept (S.E.a) 0.0021 (2.91 × 10−4) 0.0007 (2.32 × 10−4)95% Confidence limits of intercept 0.00139; 0.00273 −0.00018; 0.00088Regression coefficient (r2) 0.9999 0.9999Calculated F-value (critical F-value)b 0.729 (2.180) 1.377 (2.180)

Validation parametersSpecificity and selectivity—tCal (tCrit)c 1.34 (2.31) 1.40 (2.31)Linearity (�g mL−1) 1–20 1–24Limit of detection (�g mL−1) 0.1236 0.1307Limit of quantification (�g mL−1) 0.3746 0.3960Robustness (mean% recovery ± S.D.) 100.39 ± 1.14 100.15 ± 1.73

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rovide an additional support to the accuracy of the developedssay method, standard addition method was employed, whichnvolved the addition of different concentrations of purerug (3, 6, and 9 �g mL−1 in deionised water medium; 4, 8,nd 12 �g mL−1 in phosphate buffer medium) to a knownre-analyzed formulation sample and the total concentrationas determined using the proposed methods (n = 9) [18]. Theercent recovery of the added pure drug was calculated fromhe formula;

recovery =[

(Ct − Cs)

Ca

]× 100 (1)

here Ct is the total drug concentration measured after standardddition, Cs the drug concentration in the formulation samplend Ca is the drug concentration added to formulation (Table 3).

.5.3. PrecisionRepeatability was determined by using different levels of

rug concentrations (same concentration levels taken in accu-acy study), prepared from independent stock solutions and ana-

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able 2ccuracy and method precision data for the developed method (n = 9)

evel Predicted concentration (�g mL−1)a

Range Mean (±S.D.)

eionised waterLC (6 �g mL−1) 5.91–6.04 5.98 ± 0.043IC (14 �g mL−1) 13.89–14.24 14.06 ± 0.109HC (18 �g mL−1) 17.84–18.19 17.99 ± 0.110

hosphate buffer, pH 4.5LC (6 �g mL−1) 5.90–6.13 5.99 ± 0.064IC (13 �g mL−1) 12.83–13.28 13.09 ± 0.150HC (20 �g mL−1) 19.70–20.16 19.96 ± 0.134

a Predicted concentration of trigonelline was calculated by linear regression equatib Accuracy is given in %relative error [100 × (predicted concentration − nominal c

ficance.on paired t-test at P = 0.05 level of significance.

yzed (n = 9) (Table 2). Inter-day, intra-day and inter-instrumentariation were studied to determine intermediate precision ofhe proposed analytical methods. Different levels of drug con-entrations in triplicates were prepared three different timesn a day and studied for intra-day variation. Same procedureas followed for three different days to study inter-day vari-

tion (n = 27). One set of different levels of the concentrationsas re-analyzed using Shimadzu 1601 UV–vis spectrophotome-

er connected to computer with UV-PC software, by proposedethods to study inter-instrument variation (n = 3). The per-

ent relative standard deviation (%R.S.D.) of the predictedoncentrations from the regression equation was taken asrecision (Table 4).

Precision studies were also carried out using the real samplesf trigonelline vaginal bioadhesive tablets in a similar way totandard solution to prove the usefulness of method.

.5.4. LinearityTo establish linearity of the proposed methods, nine separate

eries of solutions of trigonelline (1–20 �g mL−1 in deionised

Mean% recovery (±S.D.) Accuracyb (%)

%R.S.D.

0.72 99.69 ± 0.715 −0.310.78 100.39 ± 0.778 0.390.61 99.94 ± 0.609 −0.06

1.06 99.87 ± 1.059 −0.131.15 100.73 ± 1.156 0.730.67 99.79 ± 0.668 −0.21

on.oncentration)/nominal concentration].

S. Chopra et al. / Spectrochimica Acta Part A 68 (2007) 516–522 519

Table 3Standard addition method for accuracy (n = 9)

Method Drug in formulation(�g mL−1)

Pure drug added(�g mL−1)

Total drug found(�g mL−1) (±S.D.)

%Analytical recovery(±S.D.)

Deionised water 10.0 3 13.04 ± 0.108 100.30 ± 0.83310.0 6 16.10 ± 0.080 100.60 ± 0.50210.0 9 18.99 ± 0.153 99.92 ± 0.807

Phosphate buffer, pH 4.5 10.0 4 13.97 ± 0.152 99.80 ± 1.08210.0 8 18.03 ± 0.127 100.14 ± 0.708

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ater and 1–24 �g mL−1 in phosphate buffer medium) wererepared from the stock solutions and analyzed. Least squareegression analysis was done for the obtained data. One-wayNOVA test was performed based on the absorbance values,bserved for each pure drug concentration during the replicateeasurement of the standard solutions (Table 1).

.5.5. Limit of detection (LOD) and limit of quantitationLOQ)

The LOD and LOQ of trigonelline by the proposed methodsere determined using calibration standards. LOD and LOQere calculated as 3.3σ/S and 10σ/S, respectively, where S is

he slope of the calibration curve and σ is the standard deviationf y-intercept of regression equation (n = 9) [13] (Table 1).

.5.6. RobustnessRobustness of the proposed methods was determined by (a)

hanging pH of the media by ±0.1 units and (b) stability of therigonelline in the both selected medium at room temperature for4 h. Three different concentrations (LC, IC and HC) were pre-ared in both the media with different pH and mean percentageecovery was determined (Table 1).

.6. Estimation from formulations

.6.1. Bioadhesive gum based vaginal gelsAn Accurately weighed quantity of gel equivalent to about

00 �g of trigonelline, i.e. 8.5 g of gel was extracted with 25 mLethanol by sonication for 30 min. This extract was centrifuged

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oncentration Intra-day repeatability %R.S.D. (n = 9)

Day 1 Day 2 Day 3

eionised water5 1.417 (0.899) 0.910 (1.022) 0.972 (1.212

10 0.805 (1.064) 0.655 (0.788) 1.070 (0.71915 1.136 (1.006) 0.787 (0.571) 0.629 (0.825

hosphate buffer, pH 4.56 1.246 (0.942) 1.064 (0.856) 0.819 (0.577

14 0.972 (0.673) 0.571 (0.832) 0.726 (1.10922 0.938 (1.118) 1.020 (1.126) 0.955 (1.314

alues in parenthesis shows the values of %R.S.D. for real samples of trigonelline va

22.03 ± 0.224 100.25 ± 1.017

t 12,000 rpm for 15 min at 4 ◦C. The supernatant was filterednd the filtrate was dried to constant weight at room temper-ture. The residue was re-dissolved in 5 mL of methanol anduitably diluted to prepare a 5 �g mL−1 concentration in bothhe media separately. The samples were then analyzed usingroposed analytical methods (Table 5).

.6.2. Bioadhesive polymer based vaginal gelsAn accurately weighed quantity of gel equivalent to about

00 �g of trigonelline, i.e. 8.5 g of gel was extracted with0 mL methanol–isopropyl alcohol (1:1, v/v) by sonication for0 min. This extract was centrifuged at 12,000 rpm for 15 mint 4 ◦C. The supernatant was filtered and the filtrate was driedo constant weight at room temperature. The residue was re-issolved in 5 mL of methanol and suitably diluted to prepare5 �g mL−1 concentration in both the media separately. The

amples were then analyzed using proposed analytical methodsTable 5).

.6.3. Bioadhesive vaginal tabletsTwenty tablets were weighed and pulverized. Amount of the

owder mixture equivalent to 100 �g of trigonelline was takennd extracted with both media separately for 30 min. These solu-ions were filtered through Whatman filter paper number 40

oncentration in both the media separately and the samplesere analyzed using proposed analytical methods (Table 5). The

esults obtained by the two methods were compared statisticallyy applying Student’s t-test and F-test.

Inter-day repeatability%R.S.D. (n = 27)

Intra-instrument repeatability%R.S.D. (n = 6)

) 1.106 (0.748) 0.661 (0.444)) 0.843 (0.563) 0.782 (0.737)) 0.851 (1.314) 1.335 (0.981)

) 1.043 (0.891) 1.419 (0.893)) 0.756 (0.968) 1.018 (1.465)) 0.971 (1.453) 0.593 (1.003)

ginal bioadhesive tablets.

520 S. Chopra et al. / Spectrochimica Acta Part A 68 (2007) 516–522

Table 5Application of spectrophotometric method to the determination of trigonelline from pharmaceutical dosage forms (n = 9)

Dosage forms Deionised water Phosphate buffer, pH 4.5

Amount foundb %Assay Amount foundb %Assay

Gum based vaginal gels (10.5 �g g−1)Mean ± S.D. (�g g−1) 10.48 ± 0.12 99.85 ± 1.14 10.54 ± 0.14 100.40 ± 1.29ta 1.15 (2.31)Fa 1.55 (2.36)

Polymer based vaginal gels (10.5 �g g−1)Mean ± S.D. (�g g−1) 10.49 ± 0.11 99.97 ± 1.05 10.57 ± 0.16 100.66 ± 1.48ta 1.59 (2.31)Fa 0.82 (2.36)

Vaginal bioadhesive tablets (525 �g)Mean ± S.D. (�g) 526.85 ± 2.71 100.35 ± 0.52 521.82 ± 2.98 99.39 ± 0.57ta 0.79 (2.31)

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. Results and discussion

In our previous research paper, we have reported a vali-ated HPTLC method for determination of trigonelline in herbalxtract and pharmaceutical dosage form [13] but the devel-pment of simple, accurate, sensitive, more convenient, lessime-consuming and economical UV-spectrophotometric meth-ds for routine analysis of the trigonelline in pure form, theirharmaceutical formulations and in vitro dissolution studies ofaginal gels and tablet formulations is highly desirable.

For media optimization various aqueous media like 0.1NCl, acetate buffers (pH 3.6–5.8), phosphate buffers (pH.8–10.2) and 0.1N sodium hydroxide were investigated. AlmostH-independent UV absorption spectra of trigonelline werebserved for the determination of trigonelline in the proposednalytical media (Fig. 2). Addition of varying amount of theethanol–isopropyl alcohol to various aqueous media did not

mprove the sensitivity of the methods and the final decision ofsing deionised water and phosphate buffer (pH 4.5) as a mediaas based on the criteria like; sensitivity of the method, cost of

ig. 2. UV-absorption spectra of 10 �g mL−1 of trigonelline (bulk form andormulation) in deionised water and phosphate buffer (pH 4.5).

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olvents, ease of preparation and applicability of the method toissolution samples. The spectra of trigonelline in the deionisedater and phosphate buffer medium are shown in Fig. 2. Themax of trigonelline in deionised water and phosphate bufferedium were found to be 265 and 264 nm, respectively. Over-

ain absorption spectra of trigonelline at 0 and 24 h time intervalsere recorded in both the media (Fig. 3). Apparent molar absorp-

ivity of the drug was found to be 4.04 × 103 L mol−1 cm−1 ineionised water and 3.05 × 103 L mol−1 cm−1 in the phosphateuffer medium.

Sandell’s index represents the number of micrograms oranograms of the determinand per millilitre of a solution havingn absorbance of 0.001 for the cell path length of 1 cm and is auitable parameter for expressing and comparing the sensitivitiesf developed UV-spectrophotometric methods [19]. Sandell’sensitivity coefficient of trigonelline was found to be 42.2 and6.7 ng cm−2/0.001A in deionised water and phosphate buffer

edia, respectively (Table 1).Two medias viz. deionised water and phosphate buffer (pH

.5), were selected for the present analytical method develop-

ig. 3. UV-absorption spectra of 10 �g mL−1 of trigonelline in deionised waternd phosphate buffer (pH 4.5) at 0 and 24 h time interval.

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ent and validation study to widen the scope of the applicabilityf method for the determination of trigonelline from differentioadhesive formulations including bioadhesive vaginal tablets,olymer- and gum-based vaginal gels. Since the actual aimf any in vitro analytical method is to simulate the in vivoonditions and minimize the interference of other excipientsresent in the formulation or dosage form, one may prefer tose phosphate buffer (pH 4.5) method for polymer-based andum-based vaginal gels, where the vaginal fluid pH is about 4.5r deionised water for bioadhesive vaginal tablets in which theablets excipients were almost completely soluble and there waso interference from other excipients present in the formulation.ence, two methods are developed separately to serve the pur-ose of analysis of different pharmaceutical dosage forms.

.1. Calibration curve

In deionised water and phosphate buffer media, the linearegression equations (r2 = 0.9999) obtained are given in Eqs. (2)nd (3), respectively;

absorbance at λ265

= [0.0233 × concentration in �g mL−1] + 0.0021 (2)

absorbance at λ264

= [0.0176 × concentration in �g mL−1] + 0.0007 (3)

.2. Analytical validation

.2.1. Specificity and selectivityThe UV-spectrum of trigonelline was not changed in the

resence of common excipients, used in the formulation ofioadhesive vaginal gels and tablets, in both the selected media.bsorption spectrum of pure drug sample was matching with

he formulation samples in both the selected media (Fig. 2).he calculated t-values were found to be less than that of the

abulated t-values, indicating that statistically there was no sig-ificant difference between the mean absorbance of solutionsrepared from pure drug sample and the formulation samplesTable 1). Therefore, proposed analytical methods are specificnd selective for the drug.

.2.2. AccuracyAccuracy ranged from −0.31 to 0.39, and −0.21 to 0.73

n deionised water and phosphate buffer media, respectivelyTable 2). The excellent mean %recovery values, close to00%, and their low standard deviation values (S.D. < 1.5)epresent high accuracy of the analytical methods. The validitynd reliability of the proposed methods was further assessedy recovery studies by standard addition method (Table 3). Ineionised water, the mean percentage recoveries (%R.S.D.) for

ower, intermediate and higher concentrations were found to be00.30 (0.833), 100.60 (0.502) and 99.92 (0.807), respectively.n phosphate buffer, the mean percentage recoveries (%R.S.D.)or lower, intermediate and higher concentrations were found

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ta Part A 68 (2007) 516–522 521

o be 99.80 (1.082), 100.14 (0.708) and 100.25 (1.017),espectively. These results revealed that any small change inhe drug concentration in the solutions could be accuratelyetermined by the proposed analytical methods.

.2.3. PrecisionPrecision was determined by studying the repeatability and

ntermediate precision. Repeatability (%R.S.D.) ranged from.61 to 0.78 and 0.67 to 1.15 in deionised water and phosphateuffer medium, respectively, at all three levels of trigonellineoncentrations (Table 2). Repeatability results indicated therecision under the same operating conditions over a shortnterval of time and inter-assay precision. Intermediate precisionxpresses within-laboratory variations in different days and inifferent instruments. In intermediate precision study, %R.S.D.alues were not more than 1.50 in all the cases (Table 4)..S.D. values found for both the analytical methods were wellithin the acceptable range indicating that these methods have

xcellent repeatability and intermediate precision. %R.S.D.alues for precision studies with real samples of trigonellineablets were found to be less than 2.

.2.4. LinearityThe linearity range for trigonelline estimation was found to be

–20 �g mL−1 (r2 = 0.9999) and 1–24 �g mL−1 (r2 = 0.9999)n deionised water and phosphate buffer medium, respectivelyTable 1). Lower values of parameters like standard error (S.E.)f slope and intercept (Table 1) indicated high precision ofhe proposed methods. Also, the mean slope and interceptalues are within the 95% confidence interval. Goodness of fitf the regression equations was supported by high regressionoefficient values and lower calculated F-values (Table 1).

.2.5. LOQ and LODIn deionised water, LOD and LOQ were found to be 0.1236

nd 0.3746 �g mL−1; and, in phosphate buffer the LOD andOQ were found to be 0.1307 and 0.3960 �g mL−1, respec-

ively, for trigonelline.

.2.6. RobustnessVariation of pH of the selected media by ±0.1 did not have

ny significant effect on the absorbance value of the trigonelline.he mean %recovery (±S.D.) values were found to be 100.39±1.14) and 100.15 (±1.73) in deionised water and phosphateuffer media, respectively (Table 1). The trigonelline solution inelected media exhibited no spectrophotometric changes over aeriod of 24 h, when kept at room temperature (Fig. 3).

.3. Estimation of formulations

In deionised water, the assay values of trigonelline for differ-nt formulations ranged from 99.85 to 100.35% with standardeviation not more than 1.14%. In phosphate buffer medium

he assay values of trigonelline for different formulationsanged from 99.39 to 100.66% with standard deviation notore than 1.48%. Assay values of formulations were same asentioned in the label claim indicating that the interference of

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xcipient matrix is insignificant in estimation of trigonelline byroposed methods. The estimated drug content with low valuesf standard deviation established the precision of the proposedethods. The results obtained from the two analytical methodsere compared statistically (Table 5). The calculated Student’s

-values and F-values did not exceed the tabulated values (for 8egrees of freedom) indicating no significant difference betweenhe methods, as far as accuracy and precision are concerned.

. Conclusion

The proposed analytical methods were simple, rapid,ccurate, precise and inexpensive and can be used for routinenalysis of trigonelline in bulk, pharmaceutical formulationsnd for dissolution samples of formulations. The sampleecoveries from all formulations were in good agreement withheir respective label claims, which suggested non-interferencef formulations excipients in the estimation. Moreover, theresent method is fast with respect to analysis time as comparedo more sophisticated chromatographic techniques.

eferences

[1] H. Vasudevan, The Science Creative Quarterly, 2006 http://www.scq.ubc.

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