Levofloxacin Hemihydrate

PURPOSE:-

To describe Raw material testing procedure of Levofloxacin Hemihydrate.

SCOPE:-

This procedure pertains to testing of Levofloxacin Hemihydrate.

370.38 g/mol

IUPAC NAME:-

(3S)-9-Fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid hemihydrate

RESPONSIBILITY:-

It is the responsibility of Analyst to follow this procedure while performing testing.

It is responsibility of Supervisor to make certain that this procedure is followed in its entirety.

It is responsibility of Manager to review and make changes where applicable.

HPLC

FTIR

Muffle Furnace

L.O.D Apparatus

MATERIALS / CHEMICALS:-

Dimethyl sulfoxide

Acetic Acid

Distilled Water

Acetone

Methanol

Glycerin

Octanol

Cupric Sulfate Pentahydrate

L-Ammonium Acetate

L-Isoleucine in Water

Methanol

DESCRIPTION (USP)

Light yellowish white to yellow white crystals or powder.

CONTENTS:-

Contents are 98.0% to 102.0 %

SOLUBILITY (USP)

Sparingly soluble in water, acetone and methanol.

Soluble in Dimethyl sulfoxide and acetic acid.

Practically insoluble in Glycerin and octanol.

IDENTIFICATION BY FTIR SPECTROPHOTOMETER:-

Measure the FTIR spectra of sample and compare with standard.

The Retention time of the major peak of the sample solution corresponds to the standard solution as obtained in the assay.

Preparation Of Buffer:-

8.5 mg/Litr ammonium acetate

1.25 grm/ Litr of Cupric Sulfate Pentahydrate

1.3 grm/Litr of L-Isoleucine in water

Preparation Of Mobile Phase:

Methanol and Buffer ( 3:7 )

Preparation Of Standard Solution:

Weigh 50mg of the Levofloxacin Hemihydrate RS and transfer it into 50ml of volumetric flask. Add mobile phase in it and sonicate to dissolve. Then make the volume up to the mark. Then filter the solution.

Preparation Of Sample Solution:-

Weigh 50mg of the Levofloxacin Hemihydrate (Raw Material) and transfer it into 50ml of volumetric flask.  Add mobile phase in it and sonicate to dissolve. Then make the volume up to the mark. Then filter the solution.

Chromatographic Conditions

Column        :           4.6mm x 25cm x Packing L1

Detector       :           UV at 360nm

Column Temperature :        45°C

Flow Rate    :           0.8ml/Min

Injection Size:          25µL

CALCULATIONS:-

Calculate the Percentage of  Levofloxacin Hemihydrate by using following Expression.

Where

ru = Peak Response from the sample solution

rs =  Peak Response from the standard solution

Cs = Concentration of Levofloxacin Hemihydrate in th standard solution (mg/ml)

Cu =   Concentration of Levofloxacin Hemihydrate in th sample solution (mg/ml)

P= Potency of Standard

2.0%  – 3.0%

OPTICAL ROTATION:-

Solvent methanol

50mg/ml in solvent.

92° – 106° at 20°C

REFERENCE DOCUMENTS

United State Pharmacopeia, USP 41,NF 36, Volume III, Page No.2395-7. for Levofloxacin

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1.  Amoxicilline Hydrate                 2.  Vitamin-C

Nitric Acid

NITRIC ACID:

Commercially available HNO3 is an azeotrpe mixture with water. Mostly found in 68.0%. Nitric Acid is considered as strong acid.

CONTENTS:

63.0 %  m/m   to   70.0 %  m/m

Concentrated HNO3 is clear, colorless or almost colorless liquid, miscible with water

Having Density   =  1.384 g/cm3  to 1.416 g/cm3

A 10 g/L solution is strongly acid and give the reaction of nitrates

APPEARANCE:

Nitric acid is clear and not more intensely colored than reference solution

BOILING POINT:

120.5°C at 1 atm pressure

CHLORIDES:

Maximum 0.5ppm when 5 gram in 10 ml of water and 0.3 ml of silver nitrate and allowed to stand for 2 minutes protected from light any opalesense is not more intense than that of a standard prepared in the same manner using 13 ml of water, 0.5 ml of nitric acid, 0.5 ml of chloride solution and 0.3 ml of silver nitrate solution.

SILFATES:

maximum 2.0ppm

Evaporate 10.0 grams to dryness with 0.2 gram of sodium carbonate. Dissolve the Residue in 15 ml of distilled water. Prepare the standard using a mixture of 2 ml of sulphate standard solution and 13 ml of distilled water

ARSENIC:

Maximum 0.02ppm

Gently heat 50 grams with 0.5 ml of sulfuric acid until white fumes begin to evolve. To the Residue add 1 ml of a 100 g/L solution of hydroxylamine hydrochloride and dilute to 2 ml with water. Prepare the standard using 1.0 ml of arsenic standard solution

IRON:

Maximum 1.0 ppm

Dissolve the Residue from the determination of sulphated ash in 1 ml of dilute hydrochloric acid and dilute 250 ml with water and dilute 5ml of this solution to 10 ml with water

HEAVY METALS:

Maximum 2.0 ppm

Dilute 10 ml of the solution prepared for the limit test for iron to 20 ml with water. 12 ml of the solution complies with the test. Prepare the reference solution using Lead standard solution

SULFATED ASH:

Maximum 0.001%

Evaporate 100.0 grams to dryness moisten the residue with a few drops of sulfuric acid and heat to dull red.

ASSAY:

To 1.50 grams add about 50 ml of water and titrate with 1M Sodium Hydroxide using 0.1 ml of methyl red solution as indicator

1 ml of 1M Sodium Hydroxide solution is equivalent to 63.0mg of HNO3.

NITRIC ACID DILUTE:

(in dilute from it contains about 125g/L of HNO3)

Dilute 20 gram of HNO3 to 100 ml with distilled water

NITRIC ACID DILUTE R1:

Dilute 40 gram of HNO3 to 100 ml with water.

NITRIC ACID DILUTE R2:

Dilute 30 gram of HNO3 to 100 ml with water.

NITRIC ACID FUMING:

Fuming HNO3 contains 98.0% of HNO3 .It is clear, slightly yellowish liquid fuming on contact with air. Having density about 1.5 g/cm3. This grade is often used in explosive industry.

ANHYDROUS HNO3:

Anhydrous HNO3 contains 99.9% of HNO3. It contains maximum 2.0% of water and 0.5% of dissolved NO2. It is also known as White fuming nitric acid, pure nitric acid

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1. Amoxicillin Hydrate        2.  Testing of Vitamin C in Drug

Amoxicillin Hydrate

Amoxicillin

is a semisynthetic aminopenicillin antibiotic with bactericidal activity. It is used as Amixicillin Hydrate Amoxicillin binds to and inactivates penicillin-binding protein (PBP) 1A located on the inner membrane of the bacterial cell wall.

IUPAC  Name

IUPAC name of Amoxicillin Hydrate is

(2S,5R,6R)-6-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, trihydrate

Molecular Formula

C16H19N3O5S • 3H2O

419.4

≥98%

Physical Appearance:

Amoxicillin Hydrate occurs as white to light yellowish white, crystals or crystalline powder.

Solubility

It is slightly soluble in water and in methanol, and very slightly soluble in ethanol (96%).

Identification

Determine the infrared absorption spectrum of Amoxicillin Hydrate as directed in the potassium bromide disk method under Infrared Spectrophotometry , and compare the spectrum with the Reference Spectrum or the spectrum of Amoxicillin RS: both spectra exhibit similar intensities of absorption at the same wave numbers.

Optical rotation

＋290°   to   ＋315°

(0.1 g calculated on the anhydrous basis, water, 100 mL, 100 mm).

Heavy metals

To 1.0 g of Amoxicillin Hydrate add 2 mL of a solution of magnesium sulfate heptahydrate (1 in 4), mix, and heat on a water bath to dryness. Carbonize the residue by gently heating. After cooling, add 1 mL of sulfuric acid, heat carefully, then heat at 500°C – 600°C to incinerate. After cooling, add 1 mL of hydrochloric acid to the residue, and heat on a water bath to dryness. Then add 10 mL of water to the residue, and heat on a water bath to dissolve. After cooling, add ammonia TS to adjust the pH to 3 – 4, and add 2 mL of dilute acetic acid. If necessary, filter, wash the residue on the filter with 10 mL of water, transfer the filtrate and washings into a Nessler tube, add water to make 50 mL, and use this solution as the test solution. Prepare the control solution as follows:

To 2.0 mL of Standard Lead Solution add 2 mL of a solution of magnesium sulfate heptahydrate (1 in 4), then proceed in the same manner as for preparation of the test solution (not more than 20 ppm).

Water:

Not less than 11.0% and not more than 15.0%

(0.1 g, volumetric titration, direct titration).

Assay

Weigh accurately an amount of Amoxicillin Hydrate and Amoxicillin RS, equivalent to about 30 mg (potency), dissolve each in a solution of boric acid (1 in 200) to make exactly 100 mL, and use these solutions as the sample solution and standard solution. Perform the test with exactly

10 mL each of the sample solution and standard solution as directed under Liquid Chromatography according to the following conditions, and calculate the peak areas, AT and AS, of amoxicillin in each solution.

Amount [µg (potency)] of amoxicillin (C16H19N3O5S)

＝ MS × AT/AS × 1000

MS = Amount [mg (potency)] of Amoxicillin RS taken

Detector:

An ultraviolet absorption photometer (wavelength: 230 nm).

Column:

A stainless steel column 4.6 mm in inside diameter and 15 cm in length, packed with octadecylsilanized silica gel for liquid chromatography (5 µm in particle diameter).

Column temperature:

A constant temperature of about 25°C.

Mobile phase:

Dissolve 1.361 g of sodium acetate trihydrate in 750 mL of water, adjust to pH 4.5 with   acetic acid, and add water to make 1000 mL. To 950 mL of this solution add 50 mL of methanol.

Flow rate:

Adjust so that the retention time of amoxicillin is about 8 minutes.

System performance:

When the procedure is run with 10 µL of the standard solution under the above operating conditions, the number of theoretical plates of the peak of amoxicillin is not less than 2500.

System repeatability:

When the test is repeated 6 times with 10 µL of the standard solution under the above operating conditions, the relative standard deviation of the peak area of amoxicillin is not more than 1.0%.

Containers and storage Containers:

Tight containers at Room temperature

≥ 2 years

Note before using Amoxicillin Product:

Do not use this medication if you are allergic to amoxicillin or to any other penicillin antibiotic, such as ampicillin, dicloxacillin, oxacillin, penicillin, and others.

Before using amoxicillin, tell your doctor if you are allergic to cephalosporins such as Omnicef, Cefzil, Ceftin, Keflex, and others. Also tell your doctor if you have asthma, liver or kidney disease, a bleeding or blood clotting disorder, mononucleosis (also called “mono”), or any type of allergy.

Amoxicillin can make birth control pills less effective. Ask your doctor about using a non-hormone method of birth control (such as a condom, diaphragm, spermicide) to prevent pregnancy while taking this medicine. Take this medication for the full prescribed length of time. Your symptoms may improve before the infection is completely cleared. Amoxicillin will not treat a viral infection such as the common cold or flu. Do not share this medication with another person, even if they have the same symptoms you have.

Antibiotic medicines can cause diarrhea. This may happen while you are taking amoxicillin, or within a few months after you stop taking it. This may be a sign of a new  infection. If you have diarrhea that is watery or bloody, stop taking this medicine and call your doctor. Do not use anti-diarrhea medicine unless your doctor tells you to.

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1.    EDTA          2.  Diclofenac Sodium            3.   Vitamin “C”

Testing of Vitamin “C” in a drug

Here is the titration method for testing of Vitamin “C “in a supplement drug. It becomes more difficult to test Vitamin “C” in drug having Other vitamins like Vitamin “A “, Vitamin “E ” Vitamin B”, extracts like Ginkobiloba or Gensing and minerals like Zinc, or Seinite etc. So here is easy method to test the Vit. “C” in that drug.

PROCEEDURE:

In the test , 1,2-diphenol-indophenol and metaphosphoric-acetic acid are used. first of all we are going to prepare both of these.

PREPARATION OF 1,2-diphenol-indophenol

Weigh 25mg of 1,2-diphenol-indolphenol in 100ml volumetric flask. add 25 ml of water and mix the weighed quantity. Now weigh 50mg of Sodium by carbonate and add in the mixture. add more 25ml of water and dissolve the reagents by continuous stirring by any mechanical means or  electromagnetic source.  and in last make the volume upto the marks using water.

PREPARATION OF Metaphosphoric Acetic-Acid

In a 100ml volumetric flask, Add 3ml of conc. phosphoric acid and 8ml of glacial acetic acid and makeup the volume upto the mark using distilled water.

ASSAY OF VITAMIN “C”

Weigh accurately the product equivalent to 100mg of Vitamin C in 200ml volumetric flask. add 70ml of metaphosphoric acetic-acid in flask and sonicate for 30 minutes. Then makeup the volume upto the mark using distilled water. Mix the solution for 5 minutes and filter through whattman filter paper. or centrifuge some solution. take 4ml of the solution in a 100ml conical flask and add 5ml of metaphosphoric acetic acid. Now titrate with 1,2-diphenol-indophenol until rose pink color .

each ml of 1,2-diphenol-indophenol used is equivalent to 0.136mg of Vit.C.

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1. EDTA                                         2. Gentamicin Sulphate

Disodium Edetate ( EDTA )

DISODIUM EDETATE: (EDTA)

Ethylenediaminetetraacetic acid (EDTA) is the aminopolycarboxylic acid. It is the disodium salt form of edetate, a polyvalent chelating agent with anti-hypercalcemic and anti-arrhythmic properties. Edetate, a heavy metal chelating agent, forms soluble stable complexes which are readily excreted by the kidneys, thereby can be used to lower serum calcium concentrations. Disodium edetate is used as a chelating agent in a wide range of pharmaceutical preparations, including mouthwashes, ophthalmic preparations, and topical preparation, typically at concentrations between 0.005 and 0.1 % w/v. Disodium edentate is also used as a water softener as it will chelate calcium and magnesium ions present in hard water.

IUPAC NAME OF EDTA:

disodium;2-[2-[carboxylatomethyl(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate

OTHER NAME:

Disodium Edetate, EDTA disodium salt, 139-33-3, Titriplex III, Edta disodium, Disodium EDTA, Disodium salt of EDTA, Chelest B, Komplexon III, Clewat N, Disodium edathamil, Disodium edta, anhydrous, Zonon D, Dotite 2NA, Selekton B2, Tetracemate disodium, Cheladrate, Mavacid ED4, Versonol 120, Ethylenediaminetetraacetic acid disodium salt, Chelest 200, Disodium versene, Endrate disodium, Sodium versenate, Metaquest B, Kiresuto B, Veresene disodium salt, Chelaplex III, Complexon III, Diso-Tate, Chelaton III, Versene NA, Ethylenediaminetetraacetic acid, disodium salt,Triplex III, Chelaton 3

C10H14N2Na2O8

336.21 g/mol

PHYSICAL APPEARANCE:

It is white crystalline powder, having melting point 240°C and boiling point 252°C.

SOLUBILITY AND pH:

It is soluble in water having pH 5.3

MANUFACTURING PROCESS:

All of the industrially used methods of manufacture of Disodium Edetate and its salts involve the addition of formaldehyde and hydrogen cyanide or an alkali metal cyanide to an aqueous solution of Disodium Edetate. The salts, or edetates, are then formed by hydrolysis. Disodium Edetate can also be formed by heating tetrahydroxyethylethylenediamine with sodium or potassium hydroxide using a cadmium oxide catalyst. Disodium EDTA was prepared by dissolving Disodium Edetate into a hot solution that contained two equivalents of sodium hydroxide. The solution was then allowed to crystallize.

HOW TO PREPARE 0.1M DISODIUM EDETATE (EDTA) :

Dissolve 37.5g of Sodium Edetate in 500ml water. Add 100ml of 1M Sodium Hydroxide and mix. Now dilute the solution to 1000ml with water.

HOW TO STANDARDIZE 0.1M DISODIUM EDETATE(EDTA):

Dissolve 0.120g of Zinc in 4ml of Hydrochloric acid. Add dilute sodium hydroxide solution until the solution is weakly acid. Now carry out the assay of zinc by complexometry. 1ml of 0.1M Disodium Edetate is equal to 6.538mg if Zinc.

HOW TO PREPARE 0.05M DISODIUM EDETATE(EDTA):

Dissolve 18.6g of Disodium Edetate in sufficient water to produce 1000ml.

HOW TO PREPARE 0.02M DISODIUM EDETATE(EDTA):

Dissolve 7.444g of Disodium Edetate in sufficient water to produce 1000ml.

HOW TO PREPARE 0.01M DISODIUM EDETATE:

Dissolve 3.722g of Disodium Edetate in sufficient water to produce 1000ml.

Mohr’s Method of Precipitation Titration

is one of the oldest titration methods still in use – it was researched and published by Karl Friedrich Mohr in 1856.The idea behind this titration is very simple – chlorides are titrated with the silver nitrate solution in the presence of chromate anions. End point is signaled by the appearance of the red silver chromate.

Intense yellow color of chromate may make detection of first signs of formation of red silver chromate precipitation difficult. As some excess of silver must be added before precipitate starts to form, if concentration of titrant is below 0.1M, we may expect significant positive error. To correct for this error we can determine a blank, titrating a solution of the indicator potassium chromate with standard silver nitrate solution. To make result more realistic we can add small amount of chloride free calcium carbonate to the solution to imitate the white silver precipitate.
Solution during titration should be close to neutral. In low pH silver chromate solubility grows due to the protonation of chromate anions, in high pH silver starts to react with hydroxide anions, precipitating in form of AgOH and Ag2O. Both processes interfere with the determination accuracy.
Exactly the same approach can be used for determination of bromides. Other halides and pseudo halides, like I- and SCN-, behave very similarly in the solution, but their precipitate tends to adsorb chromate anions making end point detection difficult.

Reaction

Reaction taking place during titration is
Ag + Cl¯  →   AgCl (s)

Sample size

Assuming 0.1M titrant concentration and 50 mL burette, aliquot taken for titration should contain about 0.12-0.16 g chloride anion (3.5-4.5 millimoles).

End point detection

Before titration small amount of sodium or potassium chromate is added to the solution, making its slightly yellow in color. During titration, as long as chlorides are present, concentration of Ag is too low for silver chromate formation. Near equivalence point concentration of silver cations rapidly grows, allowing precipitation of intensively red silver chromate which signalls end point. See precipitation titration end point detection page for more detailed, quantitative discussion.

Solutions used

To perform titration we will need titrant – 0.1 M silver nitrate solution, indicator – potassium chromate solution, and some amount of distilled water to dilute sample.

Procedure

Pipette aliquot of chlorides solution into 250mL Erlenmeyer flask. Dilute with distilled water to about 100 mL. Add 1 mL of 5% potassium chromate solution. Titrate with silver nitrate solution till the first color change.

Result calculation

According to the reaction equation

Ag Cl → AgCl

silver nitrate reacts with chloride anion on the 1:1 basis. That makes calculation especially easy – when we calculate number of moles of AgNO3 used it will be already number of moles of Cl titrated.

if you want to read Accuracy and Precision then click on the Accuracy and Precision.

Alcohol Number Determination

Alcohol Number Determination represents the number of milliliters of ethanol at 15°C obtained from 10mL of tincture or other preparations containing ethanol by the following Methods.

A: Method 1 (Distilling method)

This is a method to determine the Alcohol Number by reading the number of milliliters of ethanol distillate at 15°C obtained from 10 mL of a sample measured at 15°C by the following procedures.

1. Apparatus

Use hard glass apparatus as illustrated in figure Ground glass may be used for the joints.

1. Reagent

Alkaline phenolphthalein solution: To 1 g of phenolphthalein add 7 mL of sodium hydroxide TS and water to make 100 mL.

1. Procedure

Transfer 10 mL of the sample preparation, accurately measured at 15 ± 2°C, to the distilling flask A, add 5 mL of water and boiling chips. Distil ethanol carefully into the glass-stoppered, volumetric cylinder D. A suitable volume of distillate (mL) should be collected, according to the content of ethanol in the sample preparation. Prevent bumping during distillation by rendering the sample strongly acidic with phosphoric acid or sulfuric acid, or by adding a small amount of paraffin, beeswax or silicone resin before starting the distillation.

The figures are in mm

B: delivery tube

C: Condenser

D: Glass stoppered volumetric cylinder (25ml graduated in 0.1ml)

When the samples contain the following substances, carry out pretreatment as follows before distillation.

(i) Glycerin: Add sufficient water to the sample so that the residue in the distilling flask, after distillation, contains at least 50% of water.

(ii) Iodine: Decolorize the sample with zinc powder.

(iii) Volatile substances: Preparations containing appreciable proportions of essential oil, chloroform, diethyl ether or camphor require treatment as follows.

Mix 10 mL of the sample, accurately measured, with 10 mL of saturated sodium chloride solution in a separator, add 10 mL of petroleum benzin, and shake. Collect the separated aqueous layer. The petroleum benzin layer was extracted with two 5 mL portions of saturated sodium chloride solution. Combine the aqueous layers, and distill. According to the ethanol content in the sample, collect a volume of distillate 2 to 3 mL more than that shown

(iv) Other substances: Render preparations containing free ammonia slightly acidic with dilute sulfuric acid. If volatile acids are present, render the preparation slightly alkaline with sodium hydroxide TS, and if the preparations contain soap along with volatile substances, decompose the soap with an excess of dilute sulfuric acid before the extraction with petroleum benzin in the treatment described in (iii).To the distillate add 4 to 6 g of potassium carbonate and 1 to 2 drops of alkaline phenolphthalein solution, and shake vigorously. If the aqueous layer shows no white turbidity, agitate the distillate with additional potassium carbonate. After allowing to stand in water at 15 ± 2°C for 30 minutes, read the volume of the upper reddish ethanol layer in mL, and regard it as the Alcohol Number. If there is no clear boundary surface between these two layers, shake vigorously after addition of a few drops of water, then observe in the same manner.

B: Method 2 (Gas chromatography)

This is a method to determine the alcohol number by determining ethanol (C2H5OH) content (vol%) from a sample measured at 15°C by the following procedures.

1. Reagent

Ethanol for alcohol number: Ethanol (99.5) with determined ethanol (C2H5OH) content. The relation between specific gravity d15 of ethanol and content of ethanol (C2H5OH) is 0.797:99.46 vol% , 0.796:99.66 vol%, and 0.795:99.86 vol%.

1. Preparation of sample solution and standard solution

Sample solution: Measure accurately a volume of sample at 15 ± 2°C equivalent to about 5 mL of ethanol (C2H5OH), and add water to make exactly 50 mL. Measure accurately 25 mL of this solution, add exactly 10 mL of the internal standard solution, and add water to make 100 mL.

Standard solution: Measure accurately 5 mL of ethanol for alcohol number at the same temperature as the sample, and add water to make exactly 50 mL. Measure accurately 25 mL of this solution, add exactly 10 mL of the internal standard solution, and add water to make 100 mL.

1. Procedure

Place 25 mL each of the sample solution and the standard solution in a 100-mL, narrow-mouthed, cylindrical glass bottle sealed tightly with a rubber closure and aluminum band, immerse the bottle up to the neck in water, allowed to stand at room temperature for more than 1 hour in a room with little change in temperature, shake gently so as not to splash the solution on the closure, and allow to stand for 30 minutes. Perform the test with 1 mL each of the gas in the bottle with a syringe according to the Gas Chromatography

Under the following conditions, and calculate the ratios, QT and QS, of the peak height of ethanol to that of the internal standard.

$Alcohol&space;Number&space;=&space;\frac{Qt}{Qs}\times&space;\frac{5mL}{a&space;volume&space;ml&space;of&space;sample}\times&space;\frac{ethanol&space;content(volume%)}{9.406}$

Internal standard solution—A solution of acetonitrile (3 in 50).

Operating conditions

Detector:  A hydrogen flame-ionization detector.

Column:  A glass tube about 3mm in inside diameter and about 1.5 m in length, packed with 150- to 180µm porous ethylvinylbenzene-divinylbenzene copolymer (mean pore size: 0.0075 µm, 500 – 600m2/g) for gas chromatography.

Column temperature: A constant temperature between 105°C and 115°C.

Carrier gas: Nitrogen.

Flow rate: Adjust so that the retention time of ethanol is 5 to 10 minutes.

Selection of column: Proceed with 1 mL of the gas obtained from the standard solution in the bottle under the above operating conditions, and calculate the resolution. Use a column giving elution of ethanol and the internal standard in this order with the resolution between these peaks being not less than 2.0.