2-piperidinecarboxamide, 1-butyl-N-(2,6-dimethylphenyl)- monohydrochloride, monohydrate  
1-Butyl-2',6'-pipecoloxylidide monohydrochloride, monohydrate. 
[14252-80-3] 
 
Bupivacaine hydrochloride contains not less than 98.5 percent and not more than 101.5 percent of C₁₈H₂₈N₂O.HCl calculated on the anhydrous basis. 

Packaging and storage: Preserve in well closed containers.

US Reference Standards: USP Bupivacaine Hydrochloride RS. 

Identification:  
A: Dissolve about 230 mg in 15 mL of water in a separator, add 1 mL of 6N ammonium hydroxide and extract with three 30 mL portions of chloroform. Evaporate the chloroform at room temperature with the aid of a stream of nitrogen, and dry the residue in vacuum. Add 2 mL of chloroform to the residue, and dissolve: the infrared absorption spectrum of this solution exhibits maxima only at the same wavelengths as that of a similar preparation of USP Bupivacaine Hydrochloride RS. 

B: Ultraviolet Absorption:  
Solution: 1 in 2000. 
Medium: 0.1 N hydrochloric acid. Absorptivities at 271 nm, calculated on the anhydrous basis, do not differ by more than 3.0%. 

C: Dissolve about 50 mg in 10 mL of water in a small separator. render alkaline with 6 N ammonium hydroxide, and extract with 10 mL of ether: the aqueous layer responds to the for Chloride 

pH: between 4.5 and 6.0, in a solution (1 in 100). 

Water: Method I: between 4.0% and 6.0%. 

Residue on ignition: not more than 0. 1 %. 

Heavy metals: Method II: not more than 0.001%. 

Residual solvents:

Alcohol standard solution- Pipet 2 mL of dehydrated alcohol into a 100-mL volumetric flask, dilute with water to volume, and mix. Transfer 2.0 mL of this solution to a 50-mL volumetric flask, dilute with water to volume, and mix. The resulting solution contains 0.08% of alcohol. 
Isopropyl alcohol standard solution.  Pipet 2 mL of isopropyl alcohol into a 1000-mL volumetric flask, dilute with water to volume and mix. Transfer 2.0 mL of this solution to a 100-mL volumetric flask, dilute with water to volume, and mix. The resulting solution contains 0.004% of isopropyl alcohol. 

Test preparation- Transfer 1.0 g of Bupivacaine Hydrochloride accurately weighed, to a 25-mL volumetric flask, dilute with water to volume, and mix. 

Chromatographic system- Under typical conditions, the instrument is equipped with a flame-ionization detector and contains a 2-m X 6-mm column containing packing S3. The injection port is maintained at a temperature of about 200º C, the column at about 175º C, the detector at about 280ºC , and nitrogen is used as the carrier gas at a flow rate of about 40 mL per minute. 

Procedure- Inject equal volumes (about 5 mL) of the Test Preparation, the Alcohol Standard Solution, and the Isopropyl Standard solution successively into the gas chromatograph. Measure the responses of the alcohol peak and the isopropyl alcohol peak in each chromatogram. Determine the percentage of alcohol taken by the formula: 

2(r_U/r_S) 

and determine the percentage of isopropyl alcohol taken by the formula: 

0.1 (r_U/r_S), 

in which r_U and r_S are the responses of the respective analytes in the Test Preparation and of the corresponding analytes in the Alcohol Standard Solution and the Isopropyl alcohol standard solution, respectively. The sum of the content of alcohol and the content of isopropyl alcohol does not exceed 2%. 

Chromatographic purity: Dissolve a suitable quantity of Bupivacaine Hydrochloride in a mixture of chloroform and isopropylamine (99:1), to obtain a Test solution containing 20.0 mg per mL. Dissolve a suitable quantity of USP Bupivacaine Hydrochloride RS, accurately weighed, in the same solvent to obtain a Standard solution containing 20.0 mg per mL. Dilute a portion of this solution quantitatively with the same solvent to obtain a Diluted standard solution having a concentration of 100 mg per mL. Apply separate 10 ml portions of the three solutions on the starting line of a suitable thin-layer chromatographic plate (see Chromatography), coated with a 0.25-mm layer of chromatographic silica gel mixture. Develop the chromatogram in a suitable chamber with a solvent system consisting of a mixture of hexane and isopropylamine (97: 3) until the solvent has moved about three-fourths of the length of the plate. Remove the plate from the chamber, mark the solvent front, and dry it in warm air.  Place the plate in a closed chamber with a dish containing 1 g of iodine in a shallow layer, and allow to remain for about 5 minutes. Remove the plate from the chamber, spray it with 7 N sulfuric acid, and examine the chromatogram: the Rf value of the principal spot from the Test solution corresponds to that of the Standard solution, and the estimated size and intensity of any other spot obtained from the Test solution does not exceed that of the principal spot obtained from the Diluted standard solution (0.5%), and the total of the estimated sizes and intensities of all of the other spots obtained from the Test solution does not exceed four times that of the principal spot obtained from the Diluted standard solution 2.0%) 

Assay: Transfer about 600 mg of Bupivacaine Hydrochloride, accurately weighed, to a 250-mL conical flask, and dissolve in 20 mL of glacial acetic acid. Add 10 mL of mercuric acetate TS and 3 drops of crystal violet TS, and titrate with 0. 1 N perchloric acid VS to a green endpoint. Perform a blank determination, and make any necessary correction. Each mL of 0.1 N perchloric acid is equivalent to 32.28 mg of C₁₈H₂₈N₂O.HCl.