The dilemma of low wavelength is mainly due to the terminal absorption, which does not produce impurities at relatively low detection wavelength, but at relatively low wavelength, many substances are absorbed to different degrees, which interferes with the characteristic absorption of substances to be tested with short wavelength. Therefore, when choosing the characteristic wavelength, try to avoid choosing the low wavelength. But in some cases, in order to better achieve the effect of analysis, we cannot but choose low wavelength, which is not the past hurdle. If the low wavelength is chosen, an exclusive test is conducted early in the development of the analytical method. This need to separate, if it is the study of dissolution, exclusive test including each medium exclusive interference test, and all accessories (including capsule shell or coating powder) exclusive interference test, if each medium and under the corresponding medium of the accessories without interference of the main peak, low wavelength measuring dissolution and dissolution curve is no problem. The exclusive test of the content is relatively simple, into a blank solvent, if there is no interference with the miscellaneous peak of the main peak to be measured, the low wavelength detection content is no problem. However, the study of related substances is more complex, not only the interference of the main peak, but also the interference of various known impurities and unknown impurities. Here, the relevant substances of the preparations are used as an example to introduce some common interference situations and coping strategies.
Just started to contact a new project in the material method development, the first to exclusive test: solvent interference test and the interference test of excipients, this simple, is the configuration sample of the solvent and prescription amount of all excipients, injection liquid chromatograph, if the solvent or all excipients to the main peak or each known impurities and unknown impurities are no interference, proves that the choice of low wavelength is no problem. But to take a step back, Low wavelength solvent or all excipients (including capsule shell or coating powder) at a fixed position, Is to determine the peak out of the solvent or total excipients, The first first need to confirm whether it interferes with the determination of the main peak, Again confirm whether it interferes with the determination of each known and unknown impurity, If there is no interference, And at this low wavelength, each known impurity and unknown impurity and the main peak can be effectively separated, To prove that there is no problem with the low-wavelength selection, When performing the impurity calculation, We can calculate it by using the deduction method, It is to deduct the solvent peak or excipients peak with a fixed relative retention time for calculation.
Recently has been working on a new project, The detection wavelength is 210nm, on this condition, Both known and unknown impurities and the main peaks, However, at positions about 1.24 and 1.30, Out of 2 miscellaneous peaks, Raw material and solvent do not peak at this position, All excipient solution and preparation solution are peak, Proved to be due to total excipients, Through the investigation of individual excipients, Identified to be caused by calcium stearate, Although replacing calcium stearate from different manufacturers, The miscellaneous peak still occurs at these 2 positions, The reference preparations also had the same miscellaneous peaks, And the pre-stability by the accelerated test, None of the two miscellaneous peaks will grow, The relative position is relatively fixed, therefore, Our strategy is to deduct the miscellaneous peaks with relative retention times of 1.24 and 1.30.
However, our biggest confusion is that at the lower wavelength, in daily detection, non-solvent and other interference caused by total excipients appear, and suddenly large and small, no regularity. Most of the reasons are given as follows:
01, the HPLC system
For the whole liquid phase system, any unit is easy to cause interference, but often the most prone problems include the following points: flow phase bottle, one-way valve, filter head, detector.
1, check valve and filter head: check valve and filter head bear the brunt is the most prone to pollution, The pollutants are divided into two kinds, One is the salts that are easily soluble in water, Another type are organic impurities easily soluble in the organic phase, My general solution is as follows: remove the check valve and the filter head, Make it completely immersed in a beaker with purified water, Ultrasound for 5 to 10 minutes, Repeat 1 to 2 times (intended to remove salts soluble in water), Then with the same operation, Soak it completely in a beaker containing an organic phase (such as methanol or acetonitrile), Ultrasound for 5 to 10 minutes, Repeat 1 to 2 times (intended to remove organic impurities easily soluble in the organic phase), And then, after turning back, Again, become completely immersed in a beaker filled with purified water, Ultrasound for 5 to 10 minutes, Repeat for 1 to 2 times, Dry or dry, install. Follow the normal process for the next step.
2. Solvent bottle: the solvent bottle containing the mobile phase keeps changing the mobile phase almost every day, so the normal flushing may not affect, but for the low wavelength project, it is easy to catch the difficult impurities in the liquid phase, resulting in abnormal and irregular impurity peak in the chromatogram. Therefore, my general practice is, after normal washing, wash the solvent bottle repeatedly with purified water after holding upside down on the table for 10~20 minutes. After repeated washing with the mobile phase, reverse the table top for 10~20 minutes to eliminate the interference caused by the solvent bottle contamination.
3. Detector: In order to prevent the pollution of the detector or the daily maintenance of the detector, I generally connect the pipe to the test without the chromatography column. Low flow rate with hot water for 30 minutes, and then connect the line connected to the detector, and rinse the pipeline with hot water for 30 minutes.
4, mobile phase: our general mobile phase use period is 3~7 days, but for low wavelength related material detection is not very friendly, for example, in the process of mobile phase placement, easy to catch organic impurities from the air, and, in summer, solvent breeding bacteria, bacteria and their metabolites tend to lead to interference peak, in this case, mobile phase is best in the new system.
5. Reagent: use higher-level HPLC reagents as far as possible, without doubt, higher purity reagents or solvent,
Higher purity, less impurities, and less interference.
02. Process interference
1. Interference of plastic preparations: this is also the biggest factor causing interference.
Such as plastic weighing spoon, plastic dropper, plastic beaker, mainly organic solvent corrosion of plastic, the impurities produced. Once done a test, from capacity bottle to pipette to into the vial, are glass material, weighing spoon is iron spoon, but still have interference peak, to use the same manufacturer reagent test solvent configuration after interference peak disappear, various reasons after investigation, found is the first configuration of solvent, in order to prevent solvent pollution, once short with plastic film cover beaker, but this process, plastic film covered with liquid and solvent surface, caused the pollution.
2. Interference of the injection system: including the injection vial, vial cap, vial pad,
Recently, another test, the exclusive solvent, no interference, but the repetitive pretest of three samples
However, it cannot be repeated, and different interference peaks were generated in different positions, and the interference peaks were also generated irregularly generated in the three consecutive control solutions. Later, the causes were investigated one by one and found that the same manufacturer was the same batch of injection vial, but it was caused by the use of different injection vial pads.First into the vial pad is reused, because into the vial cushion cleaning process, has been floating on the surface of the lotion (can not sink into the lotion), into the sample pad after repeated use, the center of the vial pad is many times, there are other pollutants attached and holes, the pollutants in holes, lead to foreign material residue, resulting in pollution.
3. Choose a large brand filter film with smaller pore size and more quality assurance. For example, the conventional pore size of the laboratory filter membrane is 0.45 µ m, and a 0.22 µ m filter membrane can be selected to filter and remove potential impurities.
In a word, a word "clean", clean, clean, all kinds of measures and measures as far as possible to avoid pollution, as far as possible
To reduce the contamination in the various steps.
