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CHAPTER 3

METHOD DEVELOPMENT AND VALIDATION

OF 60% SODIUM LACTATE BY RP – HPLC TECHNIQUE

Chapter 3

1. INTRODUCTION TO SODIUM LACTATE

Sodium lactate is the taste. It is produced by fermentation of a sugar source, such as and then, by neutralizing the resulting lactic acidthe formula NaC3H5O3.

1.1 Description

DRUGS: Sodium Lactate

STRUCTURE:

Figure – 1.

IUPAC NAME :

FORMULA :

MOLACULAR WEIGHT

SOLUBILITY :

DENSITY : 1.33

MELTING POINT :

BOILING POINT :

Sodium Lactate

INTRODUCTION TO SODIUM LACTATE

is the sodium salt of lactic acid that has a mild saline taste. It is produced by fermentation of a sugar source, such as and then, by neutralizing the resulting lactic acid[3] to create a compound

DRUGS: Sodium Lactate

. Molecular structure of Sodium Lactate

: Sodium 2-hydroxypropanoate

C3H5NaO3

MOLACULAR WEIGHT : 112.06 g mol−1

: > 1.5 g/mL

: 1.33 g/mL, [1] 1.31 g/ml (60 % syrup)

: 161–162 °C 17 °C (60 % syrup) [2]

113 °C (60 % syrup) [2]

Sodium Lactate

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that has a mild saline taste. It is produced by fermentation of a sugar source, such as corn or beets,

compound having

Molecular structure of Sodium Lactate

% syrup) [1]

Chapter 3 Sodium Lactate

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1.2Usage

As a food additive, sodium lactate has the E number E325 and is naturally a liquid product, but also is available in powder form. It acts as a preservative, acidity regulator, and bulking agent.[4]

Sodium lactate is sometimes used in shampoo products and other similar items such as liquid soaps as it is an effective humectant and moisturizer.[5]

Sodium lactate is used to treat arrhythmias caused by overdosing of class I antiarrythmics, as well as pressor sympathomimetics which can cause hypertension.[6]

It also can be given intravenously as a source of bicarbonate for preventing or controlling mild to moderate metabolic acidosis in patients with restricted oral intake (for sodium bicarbonate) whose oxidative processes are not seriously impaired. However, the use in lactic acidosis is contraindicated.[7] It can cause panic attacks in patients with existing panic disorder.[8]

1.3 Regarding milk

Despite the similarity in name, sodium lactate is not chemically similar to lactose (milk sugar) and therefore need not be restricted by those with a milk allergy.[3][9] In general, lactates such as sodium, calcium, and potassium lactate are salts derived from the neutralization of lactic acid and most commercially used lactic acids are fermented from dairy-free products such as cornstarch, potatoes, or molasses.[10]Sugar or tapioca additionally may be used. However some lactic acid is fermented from dairy products such as whey[3] and lactose.[10] Whey is made of up 6.5% solids of which 4.8% is solid lactose.[11] Waste whey typically is used to produce lactic acid when the whey itself is produced as waste during the manufacture of certain dairy products.[12] As a result, such dairy-type lactic acid generally goes back into dairy products, such as ice cream and cream cheese,[10] rather than into non-dairy products. Moreover, although the lactic-acid starter culture to ferment corn or beets may contain milk,[3] sodium lactate does not contain milk protein and need not be restricted by someone avoiding milk or those with a milk allergy.[3][9]

1.4 Pharmacology

Sodium lactate is an alkalizing agent. Lactate is slowly metabolized to bicarbonate and water. This reaction depends on the cellular oxidative activity. Under normal physiological conditions conversion of sodium lactate to bicarbonate requires about one to two hours. The bicarbonate metabolite then


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has similar actions to those of sodium bicarbonate preparations. That is, bicarbonate metabolites react with acid to produce carbon dioxide and water.

1.5 Pharmacokinetics

Sodium Lactate is used as Compound Sodium Lactate (Hartmann’s) is directly administered to the systemic circulation, the bioavailability (absorption) of the active components is complete (100%). Excess of calcium is predominantly excreted by the renal system, as in the case of potassium and sodium excretion.


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2. LITERATURE REVIEW 2.1 As per Indian Pharmacopeia, Assay of Sodium Lactate is measure accurately 10 ml, evaporate to dryness in a platinum dish and ignite very gently until completely carbonized. Boil the residue with 25.0 ml of 0.05M sulphuric acid, filter and wash thoroughly with hot water. Titrate the excess of acid in the combined filtrate and washings with 0.1M sodium hydroxide using methyl orange solution as indicator. Each ml of 0.05M sulphuric acid is equivalent to 0.01121 g of C3H5NaO3. [13] 2.2 As per USP, Assay of Sodium Lactate is weigh accurately into a suitable flask a volume of solution, equivalent to about 300 mg of Sodium Lactate add 60 ml of a 1 in 5 mixture of Acetic Anhydride in Glacial Acetic Acid, mix, and allow to stand for 20 minute. Titrate with 0.1N Perchloric Acid VS, determining the end point potentiometrically. Perform a blank determination, and make any necessary correction. Each ml of 0.1N Perchloric Acid is equivalent to 11.21 mg of C3H5NaO3.

[14] 2.3 M. Staniforth*, M. O’Hanlon, T.M. Khong have been investigated the use of analytical methodology to determine lactic acids and/or polylactides. This paper reports three methods that have been successfully developed. Firstly, static headspace gas chromatography (GC) was investigated. The results show precision values of 4–5% RSD and linearity correlation coefficients of between 0.990 and 0.999 in the concentration range 300–700 mg/ml lactic acid. The method is specific for lactic acid. Secondly, an on-column GC method is presented which minimizes sample preparation and separation of lactic acid from the polylactides. Finally, anion-exclusion high-performance liquid chromatography (HPLC) method was developed. The instrumental precision was 0.1%, and linearity correlation coefficients .0.9999 were obtained in the concentration range between 50 and 200 mg/ml lactic acid. A basic hydrolysis is used to convert the polylactides to lactic acid. Collectively, these methods form a successful approach for the determination of lactic acid in consumer healthcare products.[15] 2.4 Xiumei Geng, Sufang Zhang, QianWang, Zongbao (Kent) Zhao have been worked simultaneous separation of 19 organic acids and 10 inorganic anions has been demonstrated using ion chromatography with a high capacity anion exchange column and the suppressed conductivity detector under an auto-suppression external sulfuric acid mode. Quantitative merits of this method were examined for analysis of nine organic acids of potential significance in cell culture broth. External calibration curves for these analytic were linear with correlation coefficients exceeding 0.999, and the relative standard derivations of observed analytic concentrations were less than 3.0% in both inter- and intra-


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day evaluations of aqueous standards. Developed methodology was subsequently applied to obtain organic acid profiles of Luria–Bertani liquid media, yeast extract, peptone, and the culture broth of a mutant Escherichia coli strain. Analytic recoveries observed for triplicate analysis of LB media spiked at two concentration levels ranged from 88% to 105% with less than 7% RSD. These data demonstrate quantitative accuracy for LB media and suggest that the report method may also be applicable to complex samples such as fermentation mixture and lingo cellulose hydrolysate. [16] 2.5 As per EUROPEAN PHARMACOPOEIA 5.0, Dissolve a quantity of the substance to be examined corresponding to 75.0 mg of sodium lactate in a mixture of 10 ml of glacial acetic acid R and 20 ml of aceticanhydride R. Allow to stand for 15 min. Add 1 ml of naphtholbenzein solution R and titrate with 0.1 M perchloric acid.1 ml of 0.1 M perchloric acid is equivalent to 11.21 mg of C3H5NaO3.[17] (S)-enantiomer. Transfer a quantity of the substance to be examined corresponding to 2.50 g of sodium lactate into a 50 ml volumetric flask, dilute with about 30 ml of water R and add 5.0 g of ammonium molybdate R. Dissolve and dilute with water R to 50.0 ml. Measure the angle of optical rotation (2.2.7). [17] Calculate the percentage content of (S)-enantiomer using the expression:

50 + (24.04 x a x 5/m x 50/c) a = angle of optical rotation (absolute value), m= mass of the substance to be examined, in grams, c = percentage content of C3H5NaO3 in the substance to be examined. The complex of sodium (S)-lactate formed under these test conditions is levorotatory.


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3. AIM OF PRESENT WORK As per discussion in the literature review, UV, TLC, charring method and

HPLC methods for the determination of Sodium Lactate in pharmaceutical dosage forms. Titration method is the most commonly used method for analysis of Sodium Lactate. An extensive literature survey reveals few methods for estimation of Sodium Lactate in pharmaceutical dosage forms as well as biological fluids; however, not all of these are stability indicating. Most of the reported methods either do not include stress degradation studies or are not completely validated, and they are cumbersome, time consuming and expensive. Method validation is an essential step in drug analysis. The process confirms that the analytical procedure employed for the analysis is suitable for its intended use and shows reliability of the results produced by any method. The primary objective of the present work was thus to develop and validate a method indicating HPLC method for the assay of Sodium Lactate from its drugs form. Hence, the method is useful for routine quality control analysis and also for determination of stability.

The aim and scope of the proposed work are as under:

1. To develop suitable HPLC method for Sodium Lactate. 2. To develop simple and cheap method to drugs testing. 3. Perform the validation for the developed method. 4. To develop accurate method without manual error.


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4. EXPERIMENTAL

4.1 Materials

60% Sodium Lactate was provided by Denis chem lab Ltd. as raw material. Analytical HPLC grade Acetonitrile and Methanol was purchased from Merk Pvt. Ltd. HPLC grade Sulphuric Acid and Water were obtained from Finar laboratory.

4.2 Instrumentation

The chromatographic system used to perform development and validation of this assay method was comprised of a LC-20ATvp binary pump, a SPD-20Avp UV-VIS detector and a rheodyne manual injector model 7725i with 20µl loop (Shimadzu, Kyoto, Japan) connected to a multi-instrument data acquisition and data processing system (Class-VP 6.13 SP2, Shimadzu). 4.3 Mobile phase preparation

The mobile phase consisted of 0.1M Sulphuric Acid. To prepare 0.1M Sulphuric Acid solution, 5.4ml Sulphuric Acid were pipette out and dissolve in 1000 ml HPLC grade Water. Mobile phase was filtered through a 0.45 µm nylon membrane (Millipore Pvt. Ltd. Bangalore, India) and degassed in an ultrasonic bath (Spincotech Pvt. Ltd., Mumbai). 4.4 Diluents’ Preparation

HPLC grade Water was used as diluents. 4.5 Standard Preparation

Standard solution containing 60% Sodium Lactate was prepared by dissolving accurately about 0.1 gm in 100 mL volumetric flask by diluents [water] (standard solution).

4.6 Chromatographic Conditions

Chromatographic analysis was performed on phenomenex hypersil L17 (300mm x 7.80 mm, 8 µm- rezex RHM – monosaccharide H+) column. The mobile phase was consisted of 0.1M Sulphuric Acid. The flow rate of the mobile phase was adjusted to 1.2 mL/min and the injection volume was 10 µl. Detection was performed at 210nm.


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5. RESULT AND DISCUSSION 5.1 Development and Optimization of the HPLC Method

Proper selection of the method depends upon the nature of the sample (ionic or ionisable or neutral molecule), its molecular weight and solubility. Sodium Lactate is dissolved in polar solvent hence RP-HPLC was selected to estimate them. To develop a rugged and suitable HPLC method for the quantitative determination of Sodium Lactate, the analytical conditions were selected after testing the different parameters such as diluents, buffer, buffer concentration, organic solvent for mobile phase and mobile phase composition and other chromatographic conditions. Our preliminary trials using different composition of mobile phases consisting of water with methanol or acetonitrile, did not give good peak shape.

The mobile phase consisted of 0.1M Sulphuric Acid. To prepare 0.1M

Sulphuric Acid solution, 0.54ml Sulphuric Acid were pipette out and dissolve in 1000 ml HPLC grade Water. Mobile phase was filtered through a 0.45 µm nylon membrane (Millipore Pvt. Ltd. Bangalore, India) and degassed in an ultrasonic bath (Spincotech Pvt. Ltd., Mumbai).

By using 0.1M Sulphuric Acid in 1000 ml mobile phase composition, best peak shape was obtained. For the selection of inorganic constituent of mobile phase, Sulphuric Acid was chosen to reduce the longer retention time and to attain good peak shape.


5

5.2 Method Validation

5.2.1 Linearity

Five points calibration curve were obtained in a concentration range from 0.001-0.005 gm/ml for Sodium Lactate. The response of the drug was found to be linear in the investigation concentration range and the linear regression equation was y = 590833.5x–0.0005 with correlation coefficient 0.9999. (Figure 2) Chromatogram obtain during linearity study were shown in figure 3-7.

Figure 2: Linearity curve for Sodium Lactate Where, ----series1: Concentration in gm/ml -----series 2: Area

Calculation:

Slope:

y = mx + c

dy / dx = m

m = 2363.333 – 1181.666 / 0.004 – 0.002

m = 590833.5

1 2 3 4 5

Series1 0.001 0.002 0.003 0.004 0.005

Series2 590.833 1181.666 1772.5 2363.333 2954.166

0

500

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AR

EA


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Intersect:

y= mx + c

c= y – mx

= 590.833-(590833.5)0.001

= 590.833 -590.8335

= -0.0005

Thus, the linearity equation of Sodium Lactate is y = 590833.5x–0.0005.

Calculation of correlation coefficient:

n∑ni=1 xiyi -∑

ni=1xi∑

ni=1yi

r = -----------------------------------------------------------------------------------------------

[(n∑ni=1 xi

2-(∑ni=1 xi)

2) (n∑ni=1 yi

2-(∑ni=1 yi)

2)] 1/2

5(32.50) - (0.015) (8862.50)

= ----------------------------------------------------------------------------------

{[5(0.00005) - (0.000225)][5(19199614.04) – (78543870.80)]}1/2

162.48 – 132.93

= --------------------------------------------------------------------------

{[0.000275–0.000225][95998070.22–78543870.80]}1/2


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29.5404

= -----------------------------------

{[0.00005][17454199.42]}1/2

29.5404

= --------------------------------------

[872.709]1/2

29.5404

= --------------------------------------

29.5416

= 0.9999


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Figure - 3.Linearity study chromatogram (0.001gm/ml)

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5.2.2 LOD and LOQ

The limit of detection and limit of quantification were evaluated by serial dilutions of Sodium Lactate standard solution in order to obtain signal to noise ratio of 3:1 for LOD and 10:1 for LOQ. The LOD value for Sodium Lactate was found to be 0.00001gm/ml and the LOQ value 0.0001gm/ml. Chromatogram of LOD and LOQ study were shown in Figure 8-9.

Figure - 8.LOD study chromatogram (0.00001gm/ml)

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Figure -9.LOQ study chromatogram (0.0001gm/ml)

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5.2.3 Precision

Precision study was established by evaluating method precision and intermediate precision study. System precision was evaluated by analyzing the standard solution five times. Method precision of the analytical method was determined by analyzing six sets of sample preparation. Assay of all six replicate sample preparations was determined and mean % assay value, standard deviation; % relative standard deviation was calculated. Intermediate precision of the analytical method was determined by performing method precision on another day by another analyst under same experimental condition. Assay of all six replicate sample preparations was determined and mean % assay value, standard deviation; % relative standard deviation was calculated.

Data obtain from precision experiments are given in Table 5.1 for intraday and interday precision study for 60% Sodium Lactate. The RSD values for intraday precision study and interday precision study was < 2.0% for 60% Sodium Lactate, which confirm that the method was precise.

The result of repeatability and intermediate precision study are shown in Table 5.1. The developed method was found to be precise as the %RSD values for the repeatability and intermediate precision studies were < 0.08 % and < 0.1 %, respectively, which confirm that method was precise.

Table 5.1: Evaluation data of precision study

Set Intraday (n=6) Interday (n=6) 1 590.752 603.105 2 591.133 604.099 3 589.990 603.433 4 590.254 605.072 5 590.555 604.272 6 589.872 604.089

Mean 590.426 604.011 Standard Deviation 0.4794 0.6873

%RSD 0.0812 0.1138


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Figure - 10.Precision study chromatogram (0.001gm/ml)

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5.2.4 Accuracy

The HPLC area responses for accuracy determination are depicted in Table 5.2. The result shown that best recoveries (99.00 - 100.50 %) of the spiked drug were obtained at each added concentration, indicating that the method was accurate. Chromatogram obtain during accuracy study were shown in Figure 11-13.

Table 5.2: Evaluation data of accuracy study

Concentration in gm/ml

Amount added

concentration a

(gm/ml)

Amount found Concentrationa

(gm/ml)

% Recovery

% RSD

0.001 0.0010 0.00099 99.00 0.135 0.002 0.0019 0.00199 100.50 0.356 0.003 0.0030 0.00300 100.00 0.380

a Each value corresponds to the mean of three determinations.


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Figure - 11.Accuracy study chromatogram (0.001gm/ml)

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5.2.5 Solution stability study

Table 5.3 shows the results obtain in the solution stability study at different time intervals for test preparation. It was concluded that the test preparation solution was founds table up to 48 h at 2 - 8˚ C and ambient temperature, as during this time the result was not decrease below the minimum percentage.

Table 5.3: Evaluation data of solution stability study

Intervals % Assay for test preparation solution stored

at 2-8̊̊̊̊ CCCC

% Assay for test preparation

solution stored at ambient temperature

Initial 100.22 100.02 12 h 100.10 100.00 24 h 99.98 99.99 36 h 100.00 99.98 48 h 99.97 99.97

5.2.6 Robustness

The result of robustness study of the developed assay method was established in Table 5.4. The result shown that during all variance conditions, assay value of the test preparation solution was not affected and it was in accordance with that of actual. System suitability parameters were also found satisfactory; hence the analytical method would be concluded as robust. Chromatogram obtain during robustness study were shown in figure 14-18.

Table 5.4: Evaluation data of robustness study

Robust conditions % Assay

System suitability parameters Theoretical

plates Asymmetry

Flow 0.9 ml/min 100.9 131103.3 1.0010 Flow 1.5 ml/min 99.17 113949.2 1.0074

0.05M Acetic Acid 100.3 116738.9 1.0111 0.2M Acetic Acid 99.8 103741.4 0.9965 Column change 100.0 121252.8 1.0002


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Figure 14. Standard chromatogram (0.9 ml/min flow rate)

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Figure 15. Standard chromatogram (1.5 ml/min flow rate)

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Figure 16. Standard chromatogram (0.05M Sulphuric Acid)

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Figure 17. Standard chromatogram (0.2M Sulphuric Acid)

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Figure 18. Standard chromatogram (column change)

5.2.7 System suitability

A system suitability test of the chromatographic system was performed before each validation run. Five replicate injections of standard preparation were injected and asymmetry, theoretical plate and % RSD of peak area were determined for same. Acceptance criteria for system suitability, asymmetry not more than 2.0, Theoretical plate not less than 5000 and % RSD of peak area not more then 2.0, were full fill during all validation parameter.

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6. CALCULATION AND DATA

Calculation formula used 1. Calculation formula for % assay of 60% Sodium Lactate

% Assay = Mean Test Area/Mean Standard Area x Standard Weight/100 x

100/Test Weight x Mean Test weight/Label Claim x

Potency of Standard

2. Relative standard deviation

% RSD = Standard Deviation of Measurements/Mean value of

Measurement x 100

3. Recovery

% Recovery = Amount Found/Amount Added x 100

4. Amount Found

Amount Found (gm/ml) = Mean Test Area/Mean Standard Area x

Standard Concentration

5. Amount Added

Amount Added (gm/ml) = Weight/Volume


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Linearity Study for Analytical Method Validation of 60% Sodium Lactate

Standard weight (gm) 0.1 Standard dilution 100 Standard potency 60.12 Standard concentration (gm/ml) 0.001

Concentration in gm/ml Peak Area 0.001 590.833 0.002 1181.666 0.003 1772.500 0.004 2363.333 0.005 2954.166

Correlation co-efficient 0.9999 Slope 590833.5

Intercept -0.0005


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Precision Study for Analytical Method Validation of 60% Sodium Lactate

Standard weight (gm) 0.1 Standard dilution 100 Standard potency 60.12 Label claim (gm) 0.001 Mean test weight (gm) 0.001 Standard concentration (gm/ml) 0.001

Set Intraday (n=6) Interday (n=6) 1 590.752 603.105 2 591.133 604.099 3 589.990 603.433 4 590.254 605.072 5 590.555 604.272 6 589.872 604.089

Mean 590.426 604.011 Standard Deviation 0.4795 0.6874

%RSD 0.0812 0.1138 Standard mean Area 590.485 604.131

% Assay 99.99 99.98


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Accuracy study for Analytical Method Validation of 60% Sodium Lactate

Standard weight (gm) 0.1 Standard dilution 100 Standard potency 60.12 Label claim (gm) 0.001 Mean test weight (gm) 0.001 Standard concentration (gm/ml) 0.001

Recovery Level

Set No.

Mean Area

Value added (gm)

Value found

%Recovery

0.001gm/ml

1 590.232 0.00100 0.00099 99.00 2 590.621 0.00110 0.00110 100.00 3 589.082 0.00100 0.00099 99.00

0.002gm/ml

1 1180.292 0.00198 0.00199 100.50 2 1181.045 0.00198 0.00199 100.50 3 1180.993 0.00197 0.00197 100.00

0.003gm/ml

1 1772.500 0.00300 0.00300 100.00 2 1772.360 0.00299 0.00300 100.33 3 1772.413 0.00300 0.00300 100.00

Mean Recovery Stdv. %RSD 99.33 0.5773 0.58 100.33 0.2886 0.29 100.11 0.1905 0.19


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Robustness study for Analytical Method Validation of 60% Sodium Lactate

Flow rate at 0.9ml/min Flow rate at 1.5ml/min Replicate Standard Area Standard Area

1 632.170 520.334 2 635.145 518.220 3 633.339 519.299 4 634.237 518.237 5 633.200 519.472

Mean 633.618 519.112 Standard deviation 1.1251 0.8970

%RSD 0.177 0.172 Replicate Test Area Test Area

1 632.560 519.995 2 632.914 520.046

Mean 632.737 520.020 Standard Weight (gm) 0.0010 0.0009

Test Weight (gm) 0.0009 0.0009 Mean test weight(gm) 0.0010 0.0009

Label claim(gm) 0.0010 0.0010 % Assay 99.86 100.17


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0.05M Sulphuric Acid 0.2 M Sulphuric Acid Replicate Standard Area Standard Area

1 570.133 567.244 2 573.133 566.999 3 573.159 567.230 4 574.749 566.847 5 573.001 566.990

Mean 572.835 567.044 Standard deviation 1.6722 0.1845

%RSD 0.291 0.032 Replicate Test Area Test Area

1 572.540 567.132 2 572.639 566.995

Mean 572.589 567.063 Standard Weight (gm) 0.0010 0.0010

Test Weight (gm) 0.0010 0.0010 Mean test weight(gm) 0.0099 0.0010

Label claim(gm) 0.001 0.0010 % Assay 98.95 100.00

Column change Replicate Standard Area

1 650.111 2 652.093 3 651.870 4 650.996 5 651.553

Mean 651.325 Standard deviation 0.7938

%RSD 0.121 Replicate Test Area

1 651.200 2 651.633

Mean 651.416 Standard Weight (gm) 0.0010

Test Weight (gm) 0.0009 Mean test weight(gm) 0.0009

Label claim(gm) 0.0010 % Assay 100.01


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7. REFERENCES

1. Sodium lactate, chemblink.com 2. Safety data for sodium lactate syrup. 3. Silberberg, Barrie (2009). The Autism and ADHD Diet: A Step-by-Step

Guide to Hope and Healing by Living Gluten Free and Casein Free (GFCF) and Other Interventions. Sourcebooks, Inc. p. 119. ISBN 1-4022-1845-1.

4. "Food Additive Code Numbers". South Australian Department of

Health. August 2006. Retrieved 2012-08-16. 5. US Patent 4758599, Dawn C. Minetti, "Clear, hydroalcoholic aftershave

lotion which moisturizes, conditions, and prevents irritation", issued 1988-07-19.

6. Trevor, Anthony; Bertram Katzung, Susan Masters (2008). Katzung &

Trevor's Pharmacology Examination and Board Review (8e ed.). Go Dairy Free. p. 126. ISBN 0-07-148869-3.

7. Hospira, Inc. (November 2004). "Sodium Lactate (sodium lactate)

Injection, Solution, Concentrate". Daily Med. U.S. National Library of Medicine. Retrieved 2012-08-16.

8. Eric Hollander; Daphne Simeon (2003). Concise Guide to Anxiety

Disorders. American Psychiatric Pub. p. 1. ISBN 978-1-58562-080-7. Retrieved 13 May 2012.

9. Willitts, Alice; Deborah Carter (2007). Food allergy & your child. Class

Publishing Ltd. p. 85. ISBN 1-85959-186-8. "The following ingredients do not contain milk protein and need not be avoided by people allergic to milk: … Sodium lactates."

10. Fleming, Alisa Marie (2008). Go Dairy Free: The Guide and Cookbook

for Milk Allergies, Lactose Intolerance, and Casein-free Living. Go Dairy Free. p. 90. ISBN 0-9791286-2-5.

11. Ranken, M. D.; R. C. Kill (1997). Food industries manual. Springer.

p. 125. ISBN 0-7514-0404-7.


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12. Inamdar (2009). Biochemical Engineering: Principles and Concepts. PHI Learning Pvt. Ltd. p. 254. ISBN 81-203-3677-1.

13. Indian Pharmacopiea, 1997. 14. USP30-NF25 page 3198, Vol. No. 31(5) page 1402. 15. M. Staniforth*, M. O’Hanlon, T.M. Khong Comparative study of lactic

acid and polylactides using static headspace, gas chromatography and high-performance liquid chromatography, Journal of Chromatography A, 833 (1999) 195–208.

16. Xiumei Geng, Sufang Zhang, Qian Wang, Zongbao (Kent) Zhao,

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