1. Reagents
1.1 Methanol is analytically pure.
1.2 Phosphoric acid is analytically pure.
1.3 Chromatographic purity of acetonitrile.
1.4 Sodium dodecylsulfate and sodium 1-decanesulfonate liquid chromatography.
1.5 Standard solution Accurately weigh 0.0500g each of the thiamine hydrochloride (VB1) and pyridoxine hydrochloride (VB6) standards whose moisture has been determined, dissolve in water, and accurately prepare a 50ml solution. This solution is standard solution A. Accurately weigh 0.0400g of the nicotinamide and nicotinic acid standard with the determined moisture content, dissolve in water, and accurately prepare a 20ml solution. This solution is the standard solution B. Accurately weigh 0.0500g of caffeine dried at 80 ℃ for 4h, accurately add standard solution A5ml standard solution B10ml, and then add methanol: water: phosphoric acid = 100: 400: 0.5 mixed solution, and dilute to 20ml as the standard solution. ml contains VB15μg, VB65μg, niacin 20μg, niacinamide 20μg, caffeine 50μg.
2. Instruments
2.1 High-performance liquid chromatograph: with UV detector.
2.2 Ultrasonic cleaner.
2.3 Centrifuge.
3. Analysis steps
3.1 Accurately weigh a certain amount of tablet powder or capsule content in a test tube, add methanol: water: phosphoric acid = 100: 400: 0.5 mixed solution, so that the concentration is about VB1, VB60.25mg, niacin, Niacinamide 1mg. After ultrasonic extraction for 5 minutes, centrifugation was performed at 3000 rpm / min for 5 minutes, and the supernatant was filtered through a 0.45 μm filter membrane to be injected.
3.2 The liquid sample is directly diluted with a mixed solution of methanol: water: phosphoric acid = 100: 400: 0.5, mixed thoroughly, filtered through a 0.45 μm filter membrane, and then injected.
3.3 Chromatographic conditions
3.3.1 Column: TSK C18 4.6 × 150mm
3.3.2 Column temperature: room temperature
3.3.3 Detection wavelength: VB1 260nm
3.3.4 Sensitivity: 0.02AUFS
3.3.5 Mobile phase: VB1: sodium lauryl sulfate solution (5 â–¡ 530): acetonitrile: phosphoric acid = 530: 470: 1
Caffeine, niacin, nicotinamide, VB6: sodium 1-decane sulfonate solution (1.22 â–¡ 850): acetonitrile: phosphoric acid = 850: 150: 1
3.3.6 Flow rate: 1ml / min
3.3.7 Injection volume: 10μL
3.3.8 Chromatographic analysis: Measure 10μL of standard solution and sample purification liquid into the chromatograph, and determine the retention time to determine the peak area. A = × σ × h = 2.507σh = 1.064 Wh / 2h> The peak area is more quantitative than the standard.
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