INTRODUCTION:

Mecobalamin [1], one of the coenzyme forms of vitamin B (12), acts as an important cofactor in the activities of B (12)-dependent methyltransferases. Since the discovery of Mecobalamin, it has been applied mainly in the treatment of hyperhomocysteinaemia and peripheral neuropathy. However, there is still lack of a systemic review on the clinical administration of Mecobalamin and its potential mechanism. Its molecular formula is C₆₃H₉₁CoN₁₃O₁₄Pand molecular mass 344.38g/mol. Pregabalin [2-4] is (S)-3-(aminomethyl)-5-methylhexanoic acid an anticonvulsant drug used for neuropathic pain and as an adjunct therapy for partial seizures with or without secondary generalization in adults. It has also been found effective for generalized anxiety disorder and is (as of 2007) approved for this use in the European Union and Russia. It was designed as a more potent successor to gabapentin. Its molecular formula is C₈H₁₇NO₂and molecular mass 159.23g/mol. It is effective at treating some causes of chronic pain such as fibromyalgia but not others. It is considered to have a low potential for abuse, and a limited dependence liability if misused. Pregabalin is a white to off-white, crystalline solid with a pKa1 of 4.2 and a pKa2 of 10.6. It is freely soluble in water and both basic and acidic aqueous solutions. The log of the partition coefficient (n-octanol/0.05M phosphate buffer) at pH 7.4 is – 1.35.

Fig. No. 1 Chemical structure of Mecobalamin

Fig. No. 2 Chemical structure of Pregabalin

Fig. No.3 Chemical Structure of Lipoic Acid

Lipoic acid (LA) [5-8], also known as α-lipoic acid and alpha lipoic acid (ALA) is an organosulfur compound derived from octanoic acid. LA contains two sulfur atoms (at C6 and C8) connected by a disulfide bond and is thus considered to be oxidized although either sulfur atom can exist in higher oxidation states. The carbon atom at C6 is chiral and the molecule exists as two enantiomers (R)-(+)-lipoic acid (RLA) and (S)-(-)-lipoic acid (SLA) and as a racemic mixture (R/S)-lipoic acid (R/S-LA). Only the (R)-(+)-enantiomer exists in nature and is an essential cofactor of four mitochondrial enzyme complexes. Endogenously synthesized RLA is essential for aerobic metabolism. Both RLA and R/S-LA are available as over-the-counter nutritional supplements and have been used nutritionally and clinically since the 1950s for various diseases and conditions. LA appears physically as a yellow solid and structurally contains a terminal carboxylic acid and a terminal dithiolane ring. Its molecular formula is C₈H₁₄O₂S₂and molecular mass 206.33g/mol. A detailed literature survey reveals various RP-HPLC analytical methods for the quantitative estimation of Alpha lipoic acid, Pregabalin, Mecobalamin [9-18] individually in bulk, plasma and in various pharmaceutical dosage forms. RP-HPLC methods are reported for the analysis of combination of various drugs with alpha lipoic acid, similarly Pregabalin with other drugs and Mecobalamin with other drugs [19-21]. As per our detailed literature survey as on date, there are no RP-HPLC methods available for simultaneous quantitative estimation of Pregabalin, Mecobalamin and Alpha lipoic acid in any matrix either of pharmaceutical dosage forms, plasma, etc. In addition there exist no pharmacopoeial methods available for analysis of these three drugs in combination. Thus, we here report a simple, precise, accurate, linear and a convenient RP-HPLC method and validation as per ICH guidelines [29] for simultaneous quantitative estimation of Pregabalin, Mecobalamin and Alpha lipoic acid in capsules.

MATERIALS AND METHOD:

Chemicals and Reagents Used:

The following chemicals were procured for the process: Water [HPLC Grade], Methanol [HPLC Grade], Methanol [HPLC Grade], Acetonitrile [HPLC Grade] Alpha Lipoic Acid, Mecobalamin and Pregabalin [Working standards], Orthophosphoric Acid and TEA all the chemicals were procured from STANDARD SOLUTIONS and the tablets were collected from the Local market.

Apparatus and Chromatographic Conditions:

Equipment: High performance liquid chromatography equipped with Auto Sampler and DAD or UV detector.

UV/VIS spectrophotometer: LAB INDIA UV 3000⁺

pH meter: Adwa – AD 1020

Weighing machine: Afcoset ER-200A

Temperature: Ambient

Column: Symmetry C₁₈ (4.6 x 100mm, 5.0mm, Make: Thermosil) or equivalent

Phosphate Buffer: 1.0ml of TEA in 1000 ml Water [HPLC Grade] pH adjusted with Orthophosphoric Acid.

pH: 6.0

Mobile phase: Buffer: Methanol: Acetonitrile (60: 20:20v/v)

Flow rate: 0.8 ml per min

Wavelength: 210 nm

Injection volume: 20 ml

Run time: 10min.

Preparation of buffer:

The buffer solution was prepared by dissolving accurately weighed 1.0 ml of TEAand transferred into a clean and dry 1000ml volumetric flask, dissolved and diluted with 1000ml water [HPLC Grade]. The final pH of the buffer was adjusted to 6.0 by using Ortho Phosphoric Acid.

Preparation of mobile phase:

The Mobile Phase was prepared by mixing 600 ml (60%) of the above buffer, 200ml (20%) of Methanol [HPLC Grade] and 200 ml (20%) of Acetonitrile degassed in an ultrasonic water bath for 10 minutes. Then the resultant solution was filtered through 0.45 µ filter under vacuum filtration.

Diluent Preparation:

The Mobile phase was used as Diluent.

Preparation of the Alpha Lipoic Acid, Mecobalamin and Pregabalin Standard and Sample Solution:

Preparation of Stock solution:

The stock solution was prepared by weighing accurately 10mg Alpha Lipoic Acid, Mecobalamin and Pregabalin and transferred into a clean and dry 10 ml volumetric flask. About 7 ml of diluent was added and sonicated. The volume was made upto the mark with the same diluent. From the above prepared Stock solution pipette out 1.0 ml of solution and transferred into a clean and dry 10ml volumetric flask, the diluent was added upto the mark to get final concentration. Further pipette 1.0ml of Alpha Lipoic Acid, 5.25ml of Mecobalamin and 5.25ml Pregabalin into a clean and dry 10ml volumetric flask and diluent was added upto the mark. Further pipette 0.52ml of Mecobalamin and transferred into a clean and dry 10ml volumetric flask added the diluent upto the mark.

Preparation of Sample Solution:

The sample solution was prepared by weighing equivalently 10mg of Alpha Lipoic Acid, Mecobalamin and Pregabalin and transferred into a 10 ml clean and dry volumetric flask and about 7ml of diluent was added and sonicated to dissolve it completely and the volume made up to the mark with the same solvent. From the above prepared Stock solution pipette out 1.0 ml of solution and transferred into a clean and dry 10ml volumetric flask, the diluent was added upto the mark to get final concentration. Further pipette 1.0ml of Alpha Lipoic Acid, 5.25ml of Mecobalamin and 5.25ml Pregabalin into a clean and dry 10ml volumetric flask and diluent was added upto the mark. Further pipette 0.52ml of Mecobalamin and transferred into a clean and dry 10ml volumetric flask added the diluent upto the mark. The standard and sample solutions were injected five times and the peak areas were recorded. The mean and percentage relative standard deviation were calculated from the peak areas.

System Suitability [23-28]:

The Tailing factor for the peaks due to Alpha Lipoic Acid, Mecobalamin and Pregabalin in Standard solution should not be more than 1.5. The Theoretical plates for the Alpha Lipoic Acid, Mecobalamin and Pregabalin peaks in Standard solution should not be less than 2000. The system suitability of the method was checked by injecting five different preparations of the Alpha Lipoic Acid, Mecobalamin and Pregabalin standard. The parameters of system suitability were checked.

Assay calculation for Alpha Lipoic Acid, Mecobalamin and Pregabalin:

Assay % =

Where

AT = average area counts of sample preparation.

AS = average area counts of standard preparation.

WS = Weight of working standard taken in mg.

WT =Weight of test taken in mg.

DS =Dilution of standard solution

DT =Dilution of sample solution

P = Percentage purity of working standard

System Suitability Results for Alpha Lipoic Acid:

1) The Tailing factor obtained from the standard injection was 1.7.

2) The Theoretical Plates obtained from the standard injection was 2791.9.

Assay Result for Alpha Lipoic Acid:

System Suitability Results for Mecobalamin:

1) The Tailing factor obtained from the standard injection was 1.8.

2) The Theoretical Plates obtained from the standard injection was 2902.4.

Assay Result for Mecobalamin:

System Suitability Results for Pregabalin:

1) The Tailing factor obtained from the standard injection was 1.6.

2) The Theoretical Plates obtained from the standard injection was 2018.4.

Assay Result for Pregabalin:

VALIDATION DEVELOPMENT[29]

1. PRECISION:

It is a measure of degree of repeatability of an analytical method under normal operation and it is normally expressed as % of relative standard deviation (% RSD). The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits. (Table no. 1)

Table no.1: Precision result for the drug Alpha Lipoic Acid, Mecobalamin and Pregabalin

Injection	Area for Alpha Lipoic Acid	Area for Mecobalamin	Area for Pregabalin
Injection-I	711513	974358	738688
Injection-II	680028	941079	717444
Injection-III	683903	947594	718674
Injection-IV	690459	953555	725933
Injection-V	697486	963218	733941
Average	692678	955961	726936
Standard Deviation	12448.0	13118.6	9308.0
%RSD	1.80	1.37	1.28

Acceptance Criteria: The %RSD for the area of all the five injections should not be more than 2%.

2. INTERMEDIATE PRECISION/RUGGEDNESS: To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision was performed on different day by using different make column of same dimensions. The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits. (Table no. 2)

Table no.2: Ruggedness result for the drug Alpha Lipoic Acid, Mecobalamin and Pregabalin

Injection	Area for Alpha Lipoic Acid	Area for Mecobalamin	Area for Pregabalin
Injection-I	866543	1157698	873281
Injection-II	865511	1148131	872535
Injection-III	865170	1146231	870710
Injection-IV	864852	1145822	870154
Injection-V	865420	1150414	872338
Average	865499	1149659	871803
Standard Deviation	636.8	4847.7	1315.1
%RSD	0.07	0.42	0.15

Acceptance Criteria: The %RSD for the area of all the five injections should not be more than 2%.

3. ACCURACY:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and value found. The standard solution with Accuracy -50%, Accuracy -100% and Accuracy -150% were injected into chromatographic system and calculated the amount found and amount added for Alpha Lipoic Acid, Mecobalamin and Pregabalin and further calculated the individual recovery and mean recovery values. (Table no. 3)

Acceptance Criteria: The %Recovery for each level should be between 98.0 to 102.0%.

4. LINEARITY: It is the ability of the method to elicit test result that is directly proportional to analytic concentration within a given range. It is generally reported as variance of slope or regression line. It is determined by series of three to six injections of five of more standards. Different levels of solution were prepared and injected to the chromatographic system and the peak area was measured. Plotted a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and calculate the correlation coefficient. The calibration curve was represented in fig. no. 4, 5 and 6. (Table no. 4)

Acceptance Criteria: The correlation coefficient should not be less than 0.99.

5. LIMIT OF DETECTION: The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantities as an exact value.

Limit of Detection for the drugs Alpha Lipoic Acid, Mecobalamin and Pregabalin:

The lowest concentration of the sample was prepared with respect to the base line noise and measured the signal to noise ratio. Limit of detection is the lowest concentration of the substance that can be detected, not necessarily quantified by the method. (Regression statistics) The minimum concentration at which the analyte can be detected is determined from the linearity curve by applying the following formula.

Limit of detection (LOD) = 3.3

Where S – slope of the calibration curve

σ – Residual standard deviation

Calculation of S/N Ratio for Alpha Lipoic Acid:

Average Baseline Noise obtained from Blank : 42 µV

Signal Obtained from LOD solution (0.26% of target assay concentration) : 122 µV

S/N = 122/42 = 2.90

Calculation of S/N Ratio for Mecobalamin:

Average Baseline Noise obtained from Blank : 42 µV

Signal Obtained from LOD solution (0.62% of target assay concentration) : 127 µV

S/N = 127/42 = 3.02

Calculation of S/N Ratio for Pregabalin:

Average Baseline Noise obtained from Blank : 42 µV

Signal Obtained from LOD solution (0.62% of target assay concentration) : 125 µV

S/N = 125/42 = 2.9

Acceptance Criteria: The S/N Ratio value should be 3 for LOD solution.

6. LIMIT OF QUANTIFICATION: It is defined as lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy and reliability by a given method under stated experimental conditions. LOQ is expressed as a concentration at a specified signal to noise ratio.

Limit of Quantification for the drugs Alpha Lipoic Acid, Mecobalamin and Pregabalin:

The lowest concentration of the sample was prepared with respect to the base line noise and measured the signal to noise ratio. Limit of Quantification is the lowest concentration of the substance that can be estimated quantitatively. It can be determined from linearity curve by applying the following formula

Limit of Quantification (LOQ) = 10

Where S – slope of the calibration curve

σ – Residual standard deviation

Calculation of S/N Ratio for Alpha Lipoic Acid:

Average Baseline Noise obtained from Blank : 42 µV

Signal Obtained from LOD solution (0.62% of target assay concentration) : 423 µV

S/N = 423/42 = 9.95

Calculation of S/N Ratio for Mecobalamin:

Average Baseline Noise obtained from Blank : 42 µV

Signal Obtained from LOQ solution (2.0% of target assay concentration) : 421µV

S/N = 421/42= 10.0

Calculation of S/N Ratio for Pregabalin:

Average Baseline Noise obtained from Blank : 42 µV

Signal Obtained from LOQ solution (2.0% of target assay concentration) : 418µV

S/N = 418/42= 9.2

Acceptance Criteria: The S/N Ratio value should be 10 for LOQ solution.

7. ROBUSTNESS: As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition, Temperature Variation was made to evaluate the impact on the method. The standard and samples of Alpha Lipoic Acid, Mecobalamin and Pregabalin were injected by changing the conditions of chromatography. There was no significant change in the parameters like resolution, tailing factor, asymmetric factor, and plate count.

a. The flow rate was varied at 0.7 ml/min to 0.9ml/min.: The Standard solution of Alpha Lipoic Acid, Mecobalamin and Pregabalin was prepared and analysed using the varied flow rates along with method developed flow rate. On evaluation of the above results, it was concluded that the variation in flow rate does not affected the method significantly. Hence it was indicated that the method was robust even by change in the flow rate. (Table No. 5).

Table No. 5 System Suitability Results for the drugs Alpha Lipoic Acid, Mecobalamin and Pregabalin (change in flow rate)

Name of the drug	Flow Rate (ml/min.)	System Suitability Results
Name of the drug	Flow Rate (ml/min.)	USP Plate Count	USP Tailing
Mecobalamin	0.7	2922	1.82
	0.8	2902	1.80
	0.9	2880	1.8
Alpha Lipoic Acid	0.7	2785	1.6
	0.8	2791	1.7
	0.9	2502	1.5
Pregabalin	0.7	2122	2.0
	0.8	2018	1.59
	0.9	2012	2.0

b. The Organic composition in the Mobile phase was varied from 90% to 110%:

The Standard solution for the drug Alpha Lipoic Acid, Mecobalamin and Pregabalin was prepared and analysed using the varied Mobile phase composition along with the actual mobile phase composition. On evaluation of the above results, it was concluded that the variation in 10% Organic composition in the mobile phase does not affected the method significantly. Hence it was indicated that the method was robust even by change in the Mobile phase ±10. (Table no. 6)

Table No. 6 System Suitability Results for the drugs Alpha Lipoic Acid, Mecobalamin and Pregabalin (change in % composition of organic phase)

Name of the drug	Change in Organic composition in the Mobile Phase	System Suitability Results
Name of the drug	Change in Organic composition in the Mobile Phase	USP Plate Count	USP Tailing
Mecobalamin	10% Less	2769	1.5
	Actual	2791	1.71
	10% More	2728	1.7
Alpha Lipoic Acid	10% Less	2564	1.9
	Actual	2547	1.87
	10% More	2342	1.8
Pregabalin	10% Less	2054	2.0
	Actual	2018	1.61
	10% More	2001	2.1

RESULTS AND DISCUSSION:

The present work was undertaken with the aim to develop and validate a rapid and consistent RP-HPLC method development in which the peaks will be appear with short period of time as per ICH Guidelines. The proposed method was simple, fast, accurate and precise method for the Quantification of drug in the Pharmaceutical dosage form, bulk drug as well as for routine analysis in Quality control. Overall the proposed method was found to be suitable and accurate for the Quantitative determination of the drug in tablet dosage form. The method was simple, precise, accurate and sensitive and applicable for the simultaneous determination of Alpha Lipoic Acid, Mecobalamin and Pregabalin in bulk drug and in combined dosage forms. The RP-High performance liquid chromatography (RP-HPLC) methods was developed and validated for simultaneous estimation Alpha Lipoic Acid, Mecobalamin and Pregabalin in bulk drug and in combined dosage forms. The HPLC separation was achieved on a Symmetry C₁₈ (4.6 x 100mm, 5.0mm, Make: Thermosil) or equivalent in an Isocratic Mode. The mobile phase was composed of Buffer (60%) whose pH was adjusted to 6.0 by using Ortho Phosphoric Acid, Acetonitrile (20%) [HPLC Grade] and Methanol (20%) [HPLC Grade]. The flow rate was monitored at 0.8 ml per min. The wavelength was selected for the detection was 210 nm. The run time was 10min. The retention time found for the drugs Alpha Lipoic Acid, Mecobalamin and Pregabalin were 1.803 min., 2.506 min. and 6.459 min. respectively. It was represented in fig. no. 4.

Fig. No. 4 Chromatogram represents for the drug Alpha Lipoic Acid, Mecobalamin and Pregabalin (Optimized Method Development)

The Precision data for the drugs Alpha Lipoic Acid, Mecobalamin and Pregabalin were represented in Table no. 1. The %RSD for sample should be NMT 2. The %RSD for the standard solution was found to be 1.80, 1.37 and 1.28 for the drugs Alpha Lipoic Acid, Mecobalamin and Pregabalin respectively, which is within the limits hence the method was precise. When the drugs Alpha Lipoic Acid, Mecobalamin and Pregabalin were analyzed by the proposed method in the intra and inter-day (Ruggedness) variation, a low coefficient of variation was observed it was represented in Table no. 2, which shows that the developed RP-HPLC method was highly precise. The %RSD was found to be 0.07, 0.42 and 0. 15 for the drugs Alpha Lipoic Acid, Mecobalamin and Pregabalin respectively, which were within the limits. The standard solution with Accuracy -50%, Accuracy -100% and Accuracy -150% were injected into chromatographic system and calculated the amount found and amount added for Alpha Lipoic Acid, Mecobalamin and Pregabalin and further calculated the individual recovery and mean recovery values. (Table no.3) The % recovery was found to be 99.8%- 99.8% for the drug Alpha Lipoic Acid. The % recovery was found to be 99.8% - 101.3% for the drug Mecobalamin. The % recovery was found to be 99.8% - 100.87% for the drug Pregabalin. In order to test the linearity of the method, five dilutions of the working standard solutions for the drugs Alpha Lipoic Acid, Mecobalamin and Pregabalin were prepared. The linearity was established in the range of 50 to 90ppm for the drug Alpha Lipoic Acid and 0.37 to 0.67ppm for the drug Mecobalamin and 37.5 to67.5 ppm for the drug Pregabalin. The data were represented in Table no. 4. Each of the dilution was injected into the column and the Linearity Curve was represented in Fig. no.5, 6 and 7. The Correlation coefficient (R²) should not be less than 0.99. The correlation coefficient obtained was 0.99 which was in the acceptance limit.

Fig. No.5 Calibration Curve for the drug Alpha Lipoic Acid

Fig. No. 6 Calibration Curve for the drug Mecobalamin

Fig. No. 7 Calibration Curve for the drug Pregabalin

The Limit of detection and limit of quantification of the method were calculated basing on standard deviation of the response and the slope (s) of the calibration curve at approximate levels of the limit of detection and limit of quantification. The LOD for the drugs Alpha Lipoic Acid, Mecobalamin and Pregabalin were found to be 0.504µg/ml, 0.0005µg/ml, and 0.35µg/ml respectively. The LOQ for the drugs Alpha Lipoic Acid, Mecobalamin and Pregabalin were found to be 1.3µg/ml, 0.0052µg/ml and 0.866µg/ml respectively. The Signal to noise ratio should be 3 for LOD. The results obtained were within the limit. The Signal to noise ratio should be 10 for LOQ solution. The results obtained were within the limit. The Robustness of the method was found out by testing the effect of small deliberate changes in the chromatographic conditions in the chromatographic conditions and the corresponding peak areas. The factors selected for this purpose were flow rate and percentage composition variation in Buffer, Acetonitrile [HPLC Grade] and Methanol [HPLC Grade] in the mobile phase. The method was found to be robust enough that the peak area was not apparently affected by small variation in the chromatographic conditions. The system suitability parameters were within the limits and represented in Table no.5 and 6.

CONCLUSION:

Development of new analytical methods for the determination of drugs in pharmaceutical dosage is important in pharmacokinetic, toxicological biological studies. Pharmaceutical analysis occupies a pivotal role in statuary certification of drugs and their formulations either by the industry or by the regulatory authorities. In industry, the quality assurance and quality control departments play major role in bringing out a safe and effective drug or dosage form.

The current good manufacturing practices (CGMP) and the Food Drug Administration (FDA) guidelines insist for adoption of sound methods of analysis with greater sensitivity and reproducibility. Therefore, the complexity of problems encountered in pharmaceutical analysis with the importance of achieving the selectivity, speed, low cost, simplicity, sensitivity, specificity, precision and accuracy in estimation of drugs. It was concluded that the proposed new RP-HPLC method developed for the quantitative determination of Alpha Lipoic Acid, Mecobalamin and Pregabalin in bulk as well as in its formulations was simple, selective, sensitive, accurate, precise and rapid. The method was proved to be superior to most of the reported methods. The mobile phases were simple to prepare and economical. The sample recoveries in the formulation were in good agreement with their respective label claims and they suggested non-interference of formulation excipients in the estimation. Hence the method can be easily adopted as an alternative method to report routine determination of Alpha Lipoic Acid, Mecobalamin and Pregabalin depending upon the availability of chemicals and nature of other ingredients present in the sample. The method also finds use in clinical, biological and pharmacokinetic studies for the drug Alpha Lipoic Acid, Mecobalamin and Pregabalin. The method was validated as per ICH guidelines, and validation acceptance criteria were met in all cases.

FUTURE ASPECT:

The proposed method can be use in future for the clinical, biological and pharmacokinetic studies of Alpha Lipoic Acid, Mecobalamin and Pregabalin.

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Received on 01.04.2014 Modified on 20.04.2014

Asian J. Research Chem. 7(4): April 2014; Page 426-433

Drug	% Concentration	Area	Amount Added (mg)	Amount Found (mg)	% Recovery	% Mean Recovery
Alpha Lipoic Acid	50%	710900	5	4.99	99.8%	100.3%
	100%	1421890	10	9.99	99.8%
	150%	213200	15	14.98	99.8%
Mecobalamin	50%	944703	5	4.99	99.9%	100.3%
	100%	1886671	10	9.98	99.8%
	150%	2873565	15	15.2	101.3%
Pregabalin	50%	722200	5	4.99	99.8%	100.16%
	100%	1444344	10	9.98	99.8%
	150%	2189665	15	15.13	100.87%

Linearity Level	Alpha Lipoic Acid		Mecobalamin		Pregabalin
	Conc.	Area	Conc.	Area	Conc.	Area
I	50ppm	236483	0.37ppm	316512	37.5ppm	238770
II	60ppm	462611	0.45ppm	618894	45ppm	471544
III	70ppm	711513	0.52ppm	974358	52.5ppm	738688
IV	80ppm	923580	0.60ppm	1237322	60ppm	946858
V	90ppm	1150940	0.67ppm	1540528	67.5ppm	1183152
Correlation Coefficient	0.999		0.998		0.998