An Unrealistic Drift in Assay on Anhydrous Basis towards Content Limit

Indian Journal of Pharmaceutical Sciences 679 November December 2009 multiplet for nine protons in the range δ 2.70 5.50 and these could be seven protons of glucose and two protons of the C ring of a fl avanone. A singlet at δ 2.38, integrating to three protons, indicated a phenolic acetoxyl. Another singlet for second phenolic acetoxyl group was observed at δ 2.32. Four alcoholic acetoxyls at δ 2.09 (3H), 2.07(3H), 2.06 (3H), 2.04 (3H) indicated the presence of glucose in the molecule. This data suggested compound F to be 3,5,7,3',4'-pentaoxygenated fl avanone-O-glucoside.

The content limit for assay test in almost all monograms of pharmaceutical use substance in several pharmacopeias is defined on anhydrous basis.In routine analytical practices, the assay test of pharmaceutical use substance is being performed without rendering to anhydrous state.The result of assay test is termed as assay on as-is basis.The water present in a pharmaceutical use substance is not considered as an impurity and hence the result of water content test is accounted in the result of assay on as-is basis.The water is accounted in assay on as-is basis mathematically by using industryaccepted formula for assay on anhydrous basis.The industry-accepted formula is written as (assay on as-is basis×100)/(100-%water) and out come of formula is termed as assay on anhydrous basis [1] .
The basis for industry-accepted formula is a chemical mass balance method.According to chemical mass balance method, % total theoretical mass of chemical substances present in a mixture is 100.For example, the theoretical weight percentage of sodium citrate dihydrate is sum of weight percentage of sodium citrate (87.8%) and weight percentage of water content (12.2%).In industry-accepted formula, it is assumed that sum of content of % sodium citrate (AAI) and % water content (W) is equal to 100 [2] .The theoretical mass balance equation is written as AAI+W=100 (Eqn.1),where AAI is assay on as-is basis and W is water.Usually, the experimental values of AAI and W are deviated to either positive or negative side from theoretical values.The deviation of AAI and W from theoretical value is considered as error (E).The experimental value of AAI and W is denoted as AAI±E AAI and W±E W .The Eqn.1 is modified for experimental value of AAI and W as (AAI±E AAI )+(W± E W )= 100 (Eqn.2).Mathematically the path followed for propagation of error in industryaccepted formula for assay on anhydrous basis is given as (AAI×100)/(100-W)= 100±[(E AAI ±E W )×100/ (100-W)](Eqn.3).The value AoA a cannot be 100% in Eqn. 3 because the term (E AAI +E W ) is never zero.In alternate formula, assay on anhydrous is calculated by substituting 100 by 'Φ' in industry-accepted formula and 'Φ' is sum of experimental results of assay and water content tests determined.Mathematically the path followed for propagation of error to assay on anhydrous basis in alternate formula is ).The value AoA p , in Equ.4, is function of sum of errors associated with assay and water content only.
The assay on anhydrous basis calculated using industry-accepted and alternate formula is denoted as AoA a and AoA p , respectively.The drift (ΔAoA) is a deviation of AoA value from 100 i.e.ΔAoA= |100-AoA|.The ΔAoA for industry accepted and alternate formula are denoted as ΔAoA a and ΔAoA p , respectively.The relation between ΔAoA p , ΔAoA a and water is ΔAoA a =ΔAoA p × [100/(100-W)] (Eqn.5).It is clear from Eqn.3 that the unrealistic propagation of errors in AoA a calculation is not being considered in setting assay limit [3] .Almost all substances of pharmaceutical use described in pharmacopeias have water content below 30% w/w.The substances containing water from 5% to 30% is grouped in level six for simulated model-1 preparation and difference in water between two successive levels is maintained to 5%.The values of AAI and water are termed as ideal values.The simulated model-1 is designed to understand the propagation of inaccuracy error associated with AAI and W to AoA.The ideal values of AAI and W is deviated by ±1%.The constructive mode of error propagation is set by deviating (-1%) and (+1%) the ideal value of AAI and W for fi rst and second group, respectively.The destructive mode of propagation is set by deviating (+1%) of ideal value of AAI and (-1%) of ideal value W for third group or vice versa for fourth group.The values AoA p and AoA a are calculated from deviated data of AAI and W. The ideal and deviated data is given in Table 1.The content limit is assumed between 98.0% and 102.0% for all four groups.The value of AoA a, , tabulated in Table 1, has more drift toward lower or higher side of content limit of substance and it is justifi ed as ΔAoA a = ΔAoA p ×100/ (100-W).The graph of AoA versus % water has been plotted and shown in (fi g.1).
In normal analytical practice, the decision of acceptance or rejection of pharmaceutical use substance is based on AoA and its confi dence interval.The mean value of AoA with confi dence interval (CI) (i.e.AoA±CI) should completely fall in set range of content limit [4] .The simulated model-2 is prepared to understand the propagation of standard deviation error associated with AAI and W in AoA.The theoretical value of AAI and W of sodium citrate dihydrate is varied from 12.0% to 12.4% and 87.6% to 88.0%, respectively.The variation interval between two consecutive values of W and AAI is kept constant  The KF titrator, model-Mettler DL31, equipped with a dual platinum electrode and the autotitrator, model-Mettler DL67, equipped with a glass electrode were used.The water content was determined in six replicate of CA using 2.000 g and SC using 0.300 g.The method of analysis 2.5.12 was followed for water determination [5] .Loss on drying test was performed using 0.50 g at 130° for SP.The method of analysis 2.2.32 was followed for water determination of SP [6] .The assay test was performed in six replicates by using method described in European Pharmacopiea monographs of SP, SC and CA [7][8][9] .The experimental data of AAI and W were arranged in ascending order for constructive mode of error propagation.The AoA a and AoA p for each set of AAI and W were calculated.The arithmetic mean of AAI, W, AoA a and AoA p were calculated using Eqn.7 for arithmetic mean (Ā)= (x 1 +x 2 …..x i )/n (Eqn.7).The standard deviation of AAI, W, AoA a and AoA p were calculated using Eqn.8 for standard deviation (SD)= [(Σx i -Ā) 2 /n-1] 1/2 (Eqn.8).In Eqns.7 and 8 x i is individual values and n is number of replicates.The values ΔAoA a and ΔAoA p was calculated as ΔAoA=100-AoA.The AoA a ±CI a and AoA p ±CI p were calculated using Eqn.9 for confi dence interval(CI)=(t×SD)÷ (n) 1/2 (Eqn.9)where t(student factor)=2.57at 95% confi dence interval and n=6 [10] .All experimental data tabulated in Table 3  The similar trends of observations were found for SC and CA.The predicted and experimental value of extent of standard deviation error propagation to AoA a through accepted formula was comparable for SP and SC.The predicted and experimental magnitude of drift in accepted formula was comparable for SP and SC.There was no impact on drift of AoA a and it's standard deviation for CA because the value of 100/(100-0.1) was almost equals to 1.The data related to ΔAoA a , ΔAoA p , SD AoAa and SD AoAp tabulated in Table 4. Experimentally, it was proved that the extent of propagation of errors obtained by industry-accepted formula was found higher by a factor 100/(100-%water) in comparison with alternate formula.The cause of higher standard deviation and inaccuracy has been identified in industry-accepted formula.The drift and propagation of errors should be considered during setting specification limit of substances containing higher amount of water.
Isoxazoles were reported for their various biological activities [1][2][3] .The reactive intermediate chalcones involved in their synthesis also exhibit wide range of biological activities [4][5][6] .The ability of indole to exhibit antiinflammatory, antimicrobial, antifungal activities [7][8][9] prompted the selection of indole as starting compound.In the light of these interesting biological activities, it appeared of interest to synthesize some new indolyl-isoxazole derivatives and to evaluate their antibacterial and antiinflammatory activities.Indole-3-aldehyde (2) prepared using Vilsemeir Haack reaction by reacting indole (1) with substituted acetophenones (a-j) in ethanolic KOH to obtain chalcones (3a-j), which were condensed with hydroxylamine hydrochloride in presence of

Fig. 1 :
Fig. 1: Plot of % AoA verses % Water content.The plots A, B and C, D represents constructive and destructive error of propagation, respectively.■ = AoA a , ▲= AoA p