Pyridoxine Hydrochloride
(pir'' i dox' een hye'' droe klor' ide).
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C8H11NO3·HCl205.64
3,4-Pyridinedimethanol, 5-hydroxy-6-methyl-, hydrochloride;    
Pyridoxol hydrochloride    [58-56-0].
DEFINITION
Pyridoxine Hydrochloride contains NLT 98.0% and NMT 102.0% of pyridoxine hydrochloride (C8H11NO3·HCl), calculated on the dried basis.
IDENTIFICATION
•  B. Identification Tests—General, Chloride 191: Meets the requirements
ASSAY
•  Procedure
Mobile phase: Mix 10 mL of glacial acetic acid, 0.6 g of sodium 1-hexanesulfonate, and 700 mL of water in a 1000-mL volumetric flask. Adjust with glacial acetic acid or 1 N sodium hydroxide to a pH of 3.0. Add 235 mL of methanol, and dilute with water to volume.
Internal standard solution: 5 mg/mL of p-hydroxybenzoic acid in Mobile phase
Standard solution: Prepare a 0.5-mg/mL solution of USP Pyridoxine Hydrochloride RS in Mobile phase. Transfer 10.0 mL of this solution and 1.0 mL of Internal standard solution to a 100-mL volumetric flask, and dilute with Mobile phase to volume.
Sample solution: Prepare a 0.5-mg/mL solution of Pyridoxine Hydrochloride in Mobile phase. Transfer 10.0 mL of this solution and 1.0 mL of Internal standard solution to a 100-mL volumetric flask, and dilute with Mobile phase to volume.
Chromatographic system 
Mode: LC
Detector: UV 280 nm
Column: 4.6-mm × 25-cm; packing L1
Flow rate: 1.5 mL/min
Injection size: 20 µL
System suitability 
Sample: Standard solution
[Note—The relative retention times for pyridoxine and p-hydroxybenzoic acid are about 0.7 and 1.0, respectively. ]
Suitability requirements 
Resolution: NLT 2.5 between pyridoxine and p-hydroxybenzoic acid
Relative standard deviation: NMT 3.0% for the ratios of the pyridoxine peak area response to the internal standard peak area response
Analysis 
Samples: Standard solution and Sample solution
Calculate the percentage of pyridoxine hydrochloride (C8H11NO3·HCl) in the portion of Pyridoxine Hydrochloride taken:
Result = (RU/RS) × (CS/CU) × 100
RU== internal standard ratio (peak response of pyridoxine/peak response of the internal standard) from the Sample solution
RS== internal standard ratio (peak response of pyridoxine/peak response of the internal standard) from the Standard solution
CS== concentration of USP Pyridoxine Hydrochloride RS in the Standard solution (mg/mL)
CU== concentration of Pyridoxine Hydrochloride in the Sample solution (mg/mL)
Acceptance criteria: 98.0%–102.0% on the dried basis
IMPURITIES
•  Residue on Ignition 281: NMT 0.1%
•  Heavy Metals, Method II 231: NMT 30 ppm
SPECIFIC TESTS
•  Content of Chloride
Sample: 500 mg of Pyridoxine Hydrochloride
Blank:  50 mL of methanol
Titrimetric system 
Mode: Direct titration
Titrant: 0.1 N silver nitrate VS
Endpoint detection: Visual
Analysis: Dissolve the Sample in 50 mL of methanol. Add 5 mL of glacial acetic acid and 2–3 drops of eosin Y TS. Titrate with the Titrant. Perform a Blank determination.
Calculate the percentage of chloride (Cl) in the Sample taken:
Result = {[(VS VB) × N × F]/W} × 100
VS== Titrant volume consumed by the Sample (mL)
VB== Titrant volume consumed by the Blank (mL)
N== actual normalilty of the Titrant (mEq/mL)
F== equivalency factor, 35.45 mg/mEq
W== Sample weight (mg)
Acceptance criteria: 16.9%–17.6% on the dried basis
•  Loss on Drying 731: Dry a sample in vacuum over silica gel for 4 h: it loses NMT 0.5% of its weight.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight, light-resistant containers.
•  USP Reference Standards 11
USP Pyridoxine Hydrochloride RS Click to View Structure
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/QuestionContactExpert Committee
MonographHuy T. Dinh, M.S.
Scientific Liaison
1-301-816-8594
(DS2010) Monographs - Dietary Supplements
Reference StandardsRS Technical Services
1-301-816-8129
rstech@usp.org
USP35–NF30 Page 4491
Chromatographic Column— 
Chromatographic columns text is not derived from, and not part of, USP 35 or NF 30.