PA/PH/OMCL (11) 04 - OMCL Guideline on qualification of HPLC equipment (Annex 1) 在所有其它情况下采用以下程序
Solutions: 溶液
DAD: caffeine Std. 5 from the linearity testing 线性测试中咖啡因标准溶液5号 UV/VIS: 1.0 μg/ml anthracene solution in water 蒽水溶液
Settings: 设置
Mobile phase: 15% acetonitrile in water 流动相：15%乙腈水溶液
30-50 x 2,1-4.6 mm or capillary 2.0 m x 0.12 mm ID
色谱柱：RP18，5μm, 30-50×2.1-4.6mm或毛细管柱20m×0.12mm内径 Oven temperature: 40 °C 柱箱温度：40℃
Flow rate: 1.0 ml/min (adjusted by using 15% acetonitrile in water) 流速：1.0ml/min（用15%的乙腈水溶液调节） Detection: scan from 230 nm to 290 nm (DAD) 检测：自230至290nm扫描（DAD） Injection volume: 20 μl 进样体积：20μl
Method方法:
DAD: Inject 20μl of the caffeine solution and record the spectrum. The maximum is at 272 nm and the minimum at 244 nm.
DAD: 咖啡因溶液进样20μl，记录色谱图，最大吸收波长为272nm，最小为244nm.
UV/VIS: fill the cell with the anthracene solution and change the wavelength from 248 to 254 nm in 1 nm steps; record the maximum of absorption. The theoretical value is 251 nm.
UV/可见光：向池中注入蒽溶液，将波长以1nm的步距从248变化至254nm，记录最大吸收值。理论值为251nm。
Limit限度s: ≤2 nm
HPLC FLUORESCENCE DETECTOR 荧光检测器
The following three parameters are proposed for the performance of the periodic and motivated check of the HPLC fluorescent detector:
下面三个参数建议作为荧光检测器性能周期自主检查
WAVELENGTH ACCURACY EXCITATION 波长激发准确度
Method方法: 方法
Rinse and fill the measuring cell with de-ionized water 用去离子水淋洗并充满检测池
Adjust the excitation wavelength to 350 nm. 调节激发波长为350nm
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PA/PH/OMCL (11) 04 - OMCL Guideline on qualification of HPLC equipment (Annex 1) Measure the emission and subtract 397 nm (theoretical value). 测量发射波长减去397nm（理论值）
Limit限度限度s: ±3 nm
WAVELENGTH ACCURACY EMISSION 波长发射准确度
Method方法: 方法
Rinse and fill the measuring cell with de-ionized water 用去离子水淋洗并充满检测池
Adjust the emission wavelength to 397 nm. 调节发射波长为397nm
Measure the excitation and subtract 350 nm (theoretical value). 测量激发波长减去350nm（理论值）
Limit限度限度s:±3nm
SENSITIVITY 灵敏度
Solutions: 溶液
Quinine HCl.2H2O solution conc. 0.015μg/ml (=15 ppb)
二水盐酸喹啉溶液，浓度0.015μg/ml (=15 ppb)
The quinine solution is prepared with the following mobile phase: dissolve 6.8 g of potassium
dihydrogen phosphate R and 3.0 g of hexylamine R in 700 ml of water R, adjust to pH 2.8 with dilute phosphoric acid R, add 90 ml of acetonitrile R and dilute to 1000.0 ml with water R.
喹啉溶液用以下流动相制备：溶解6.8g磷酸二氢钾和3.0g己胺于700ml试剂级水中，用稀磷酸调pH至2.8，加90ml乙腈并用试剂级水稀释至1000.0ml.
Settings: 设置
The chromatographic conditions are set according to Ph. Eur. ―Quinine HCl‖ (01/2005:0018), test ―Other cinchona alkaloids‖, with modified flow rate and acetonitrile concentration. 色谱条件设定依据欧洲药典―盐酸喹啉‖（01/2005:0018），检测―金鸡纳生物碱‖，改变流速和乙腈浓度。 Mobile phase: as above 流动相：同上
Column: RP18, 5 μm, 250 × 4.6 mm 色谱柱: RP18, 5 μm, 250 ×4.6 mm Flow rate: 1.2 ml/min 流速：1.2 ml/min
Excitation wavelength: 350 nm 激发波长：350nm
Emission wavelength: 397 nm 发射波长：397nm
Flow-cell volume: 8 μl (for this example, a Waters 2475 Multi Fluorescentie detector was used. Flow-cell volume may vary depending on the instrument manufacturer)
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PA/PH/OMCL (11) 04 - OMCL Guideline on qualification of HPLC equipment (Annex 1) 流动池体积：8μl（此例中，使用的是Waters2475多荧光检测器，流动池体积可能根据仪器厂商不同而不同）
Method方法:
Inject 10μl of the quinine solution and measure the peak height. 进样喹啉溶液10μl，测量峰高度
Inject 10μl of the blank and measure the peak height of the noise. 进样空白溶液10μl，测量噪音峰高
Divide the peak height of the quinine solution by 3 times the peak height of the noise. 将喹啉溶液峰高除以3倍噪音峰高
Divide the concentration of the quinine solution by the previously obtained factor. 将喹啉溶液浓度除以之前的系数 Limit限度限度s: ≤ 0.5 ppb
HPLC ELECTROCHEMICAL DETECTOR 高效液相电化学检测器
Accuracy and stability of the signal are the proposed parameters to be tested during the periodic and motivated check of the HPLC electrochemical detector. 电化学检测器的信号准确度和稳定性是定期自主检测中需要测定的参数
ACCURACY AND STABILITY OF THE SIGNAL 信号准确度和稳定性
Settings: 设定
Cell potential of a dummy cell: 800 mV 空白池电压：800mv Rise time filter: 0.1 s 增加时间过滤：0.1s Range: 0.1 nA 范围：0.1nA
Temperature: 30 °C 温度：30℃
Method方法:
Accuracy: Measure the electric current and subtract 2.67 nA (theoretical value) 准确度：检测电流并减去2.67nA（理论值）
Stability: Measure the noise over a period of 5 minutes 稳定性：连续5分钟测量噪音
Limit限度s:
准确度（池电流）：0.05nA
Stability of the signal (Noise): max. 2 pA or 20 mV 信号（噪音）稳定性：最大2pA或20mV
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PA/PH/OMCL (11) 04 - OMCL Guideline on qualification of HPLC equipment (Annex 1) HPLC RID DETECTOR 高效液相RID检测器
Signal to Noise ratio and drift over time are the parameters proposed for the periodic and motivated check of the HPLC RID (refractive index) detector. 高效液相RID（折光率）检测器的信噪比和对时间的漂移是定期自主检测中需要测定的参数
SIGNAL TO NOISE RATIO 信噪比
Solutions溶液:
Standard solution: D-fructose concentrate solution at 4.0 mg/ml (dilute 200.0 mg fructose + 20 ml water + 25.0 ml acetonitrile up to 50.0 ml with water for HPLC)
标准溶液：D-左旋糖溶液4.0mg/ml（将200.0mg左旋糖+20ml水+25.0ml乙腈，用色谱纯水稀释至50.0ml）
Settings设置:
Column: spherisorb NH2 (or equivalent) 250 ×4.6 mm or other 色谱柱：spherisorb NH2 (或相当的) 250 ×4.6 mm 或其他 Oven temperature: 38°C 柱温箱温度：38℃ Flow rate: 1.0 ml/min 流速: 1.0 ml/min
Injection volume: 20 μl 进样量: 20 μl
Mobile phase: 0.253g sodium hydrogen phosphate R in 220 ml + 780 ml acetonitrile 流动相：0.253g磷酸氢二钠溶于220ml+780ml乙腈
Method方法:
After equilibration, inject three times a blank solution of mobile phase over a run time where the system is stable. Measure the baseline noise over an appropriate period.
平衡后，以流动相为空白进样三针，系统稳定运行适当时间，测定适当时长内噪音基线 The baseline noise is accepted if the mean height of the three replicates is < 1000 μV. 可接受基线噪音：三次平行进样平均值< 1000 μV
To calculate the signal to noise ratio, inject three times a solution of fructose at 0.4 mg/ml and calculate the mean of the three replicates.
计算信噪比，左旋糖溶液0.4mg/ml进样三针，计算平均值
Limit限度s: S/N ≥10 限度：信噪比≥10
DRIFT OVER A DEFINED PERIOD OF TIME 指定时长漂移
Method方法: 方法
Calculate the slope of the amplitude of random variations in the detector’s signal over 1 minute.
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