The development of analytical methods mainly includes the selection of chromatographic columns, the selection of mobile phases, the selection of detection wavelengths, and the optimization of gradients. At present, HPLC is mostly used in reversed phase, so this article mainly uses reversed phase as an example to explain.
The production of APIs has very strict requirements on product purity and impurity content. The chromatographic column used for testing is required to have a higher theoretical plate number, which can provide better resolution and thus have greater separation of possible impurities. Possibility, so the chromatographic column length of the 5um packing should be 250mm, and the column length of the 3.5um packing should be 150mm, basically the longest column length of each particle size. I prefer the new sub-two-micron chromatographic column in the past two years. The column length of 50mm can provide a high number of theoretical plates, and the column length and particle size are small, and the flow rate is increased a lot, which can save a lot of analysis time. , Greatly improve work efficiency. Generally, a column with a diameter of 4.6mm or 3.0mm is used, too thin may increase the extra-column effect. The pore size of the filler does not need to be considered for small molecule synthetic drugs. Common analytical columns are around 100A, which can meet the needs of analysis and detection.
For API analytical method development, it is generally required to perform column screening experiments. At least three different types of chromatographic columns should be used, three of each type, from different manufacturers.
The three types include:
1) Ordinary C18 or corresponding C8 chromatographic columns, such as Symmetry C18 or C8 from Waters, Pack Pro C18 or C8 from YMC, RX C8 from Agilent, etc. Other companies such as Philomon and Thermoelectric also have corresponding chromatographic columns;
2) End-capped or polar embedded chromatographic columns, such as Symmetry Shield RP18 or RP8 from Waters, XTerra RP18 or RP8, ODS AQ from YMC, Zorbax SB AQ from Agilent, etc. Other companies such as Philomon and Thermoelectric also have corresponding的chromatographic column;
3) Chromatographic columns with fillers modified with other functional groups, such as phenyl columns, are available in many companies.
Generally, different types of chromatographic columns will have great differences in selectivity. Different manufacturers of the same type of chromatographic columns will also have differences in selectivity. This is mainly determined by the nature of the filler and the production process. Sometimes one is used. Only chromatographic column separation is not good. In addition to optimizing the gradient and mobile phase, it is also a good choice to change a column from a manufacturer. There is no difference in selectivity between C18 and C8 for chromatographic columns of the same brand and model, but the retention of C18 is stronger and the resolution of the same sample is higher. We generally prefer to use C18. When selecting chromatographic columns, we try our best to choose the top manufacturers in the industry. The quality of the columns is good, and a lot of effort can be saved when developing analytical methods, and the analytical methods produced are also guaranteed. A drug may last more than ten years or more from development to market. The manufacturer has the strength, and the column selected during the development of the method will be purchased after a few years when needed, and the repeatability of the analysis can also be guaranteed. Using several chromatographic columns for screening and optimization of analysis methods can improve the quality of the analysis methods as much as possible and ensure the credibility of the detection results.
The columns I prefer to use are: Agilent’s Zorbax Eclipse XDB-C18, Zorbax Eclipse Plus C18, Waters’ Symmetry C18, XTerra RP18, XTerra MS C18, etc. YMC’s columns are sometimes a good alternative, Philomenon’s columns Philomon’s pillars have a high market share at home and abroad, but I feel that the pressure of the pillars is high and the service life is short.
Column selection requirements for preparation analysis methods are basically the same as those for API. For intermediates and the columns used in the development of reaction tracking (IPC) analysis methods in the production process, we generally select one or two common C18 or capped columns directly according to the nature of the sample. The treated column simplifies the screening process.
The solvents commonly used as reversed-phase mobile phases are methanol and acetonitrile. Methanol has its cost-effective advantages, but methanol has high activity and may react with some samples. In addition, methanol has ultraviolet absorption at low wavelengths, which will reduce the sensitivity of the analytical method; Although acetonitrile is expensive and more toxic than methanol, it has stronger elution capacity than methanol and rarely reacts with samples. The pressure of the mobile phase system is much lower than that of methanol, and the cut-off wavelength is 20nm lower than that of methanol, which increases the detection Impurities that can be absorbed only at low wavelengths, so we generally tend to use more acetonitrile and less methanol. But sometimes the sample peak shape is not good or the separation is not good. It is a good choice to change the solvent. After all, different solvents provide different selectivity.
The optimization of the mobile phase is mainly done in the water phase. Acid, alkali, and salt can be added to the water to improve the peak shape and increase the resolution. Alkali is rarely added to the mobile phase, and acid is mainly added. Commonly used acids include phosphoric acid, trifluoroacetic acid, formic acid, acetic acid, perchloric acid, methanesulfonic acid, etc., among which phosphoric acid and trifluoroacetic acid, phosphoric acid are the most commonly used. There is no ultraviolet absorption at low wavelengths, while trifluoroacetic acid has at low wavelengths, but trifluoroacetic acid is volatile and phosphoric acid is not good, so it is purely liquid phase, phosphoric acid is most suitable at low wavelengths, trifluoroacetic acid has absorption, and when running gradients The baseline drift is very serious, and trifluoroacetic acid should be considered as the first choice for liquid quality. In recent years, the addition of formic acid or acetic acid is more popular. Under normal circumstances, there is not much difference between these kinds of acids. We are more considering changing the pH value of the mobile phase by adding acid to improve the resolution and peak shape of the sample. The following two figures show the effect of the pH change of the mobile phase on the separation and peak shape of a set of samples.
It can be seen from the figure:
1) Changing the pH of the mobile phase can change the retention time and resolution of the sample;
2) Changing the pH value of the mobile phase can even change the peak sequence of some samples;
3) Changing the pH of the mobile phase can change the peak height of the sample, that is, adjust the peak shape.
The higher the peak of the sample with the same injection volume, the better the peak shape. It can be seen from the figure that most samples have better peak shapes than neutral at low pH values. This is mainly determined by the nature of the chromatographic column itself. Chromatographic columns are mainly silica gel matrix. The existing filler treatment process cannot remove all the residual silanols on silica gel. Silanos will cause sample peak tailing. It is generally believed that the pKa of silanos is between 3.5 and 4.5, and the pH value is low. It can help inhibit the activity of silanol and reduce tailing, thereby improving peak shape and improving resolution. Adding 0.1% (volume) of phosphoric acid or trifluoroacetic acid to the aqueous solution has a pH of about 2. It is used as a mobile phase to inhibit the activity of silanols. Therefore, when developing a liquid phase analysis method, the first choice is water plus 01.% phosphoric acid. , And then optimize based on this.
When using acid alone is not enough, you should consider using buffer salts. The selection principle of buffer salts is: simple, stable, strong buffering capacity, simple preparation, and corresponding acid or alkali should be used when pH value needs to be adjusted. The commonly used buffer salts are phosphate, mainly potassium and sodium salts, and then acetate. The commonly used salt concentration is about 10-20 mM. In the past, because of the production process of the chromatographic column packing, it was often necessary to add triethylamine in the mobile phase to reduce tailing, but triethylamine has a great impact on the life of the chromatographic column, and now new chromatographic columns are no longer needed. The mobile phase sometimes needs to adjust the pH value to alkaline. The specific pH depends on the tolerance range of the chromatographic column. NaOH, KOH solution or ammonia water are commonly used as reagents to adjust the alkaline pH of the buffer salt solution, or it can be in water Separately add ammonia water as an alkaline mobile phase.
When the buffer salt is used as the mobile phase, if the peaks are too early, the peak shape is very poor, and the compound peaks of similar structure cannot achieve baseline separation because of tailing or the peak shape is too wide, you can consider using ion pair reagents, commonly used ion pair reagents Mainly various sodium alkyl sulfonates and tetrabutylammonium salts, but when ion-pairing reagents are used in the mobile phase, the system needs a long equilibration time and the sample retention time is not very stable, because the background absorption baseline of the ion-pairing reagents will be very high. Poor, and it takes a long time to clean after finishing the sample, so we try not to use ion pair reagents.
When using buffer salts, pay attention to the problem of possible salt precipitation after the mobile phase is mixed and the problem of serious baseline drift caused by salt background absorption. Especially after adding ammonium acetate to the mobile phase, the baseline drops very seriously when the gradient changes at low wavelengths. To affect the accurate quantification of small impurities, you can consider adding 10% water to acetonitrile, and adding buffer salt 10 times the concentration of the aqueous solution in advance, so that the concentration of the two salts A and B in the gradient is the same, which can avoid serious baseline drift The problem.
The general structure of the bulk drug is relatively large and the molecular structure is relatively complex. The effect of adding phosphoric acid to water when developing analytical methods may not be effective. It is usually required to try at least two phosphate buffer solutions with pH values of 2 and 6.5, and perform the mobile phase based on the results. pH optimization, if the effect is not ideal, then try other buffer salt solutions. When developing analytical methods for intermediates or IPC, the mobile phase selection process can be simplified as appropriate based on experience.
Gradient optimization is mainly to adjust the retention time of the sample by adjusting the initial ratio of the mobile phase and the slope of the gradient to optimize the resolution of the sample. The figure below shows the effect of the change in the initial ratio of the organic phase in the gradient on the separation of a set of samples.
It can be seen from the figure that the smaller the initial ratio of the organic phase, the longer the retention time of the sample. As the gradient changes, the order of the peaks of the sample may also change. When we do gradient optimization, we mainly adjust the starting ratio and slope of the gradient. Current chromatographic columns either use a new end-capping process, or have embedded polar groups, and have relatively high water resistance. Many compounds exist in the form of ions under acidic conditions, with greater polarity. In order to improve the resolution of the sample, try to use a large proportion of water as the starting point of the gradient. For the mobile phase with buffer salt added, it should be noted that there should be no salt precipitation when the composition of the mobile phase changes during the gradient change.
For the mobile phase of water plus 0.1% phosphoric acid, you can start with 95% water at the beginning and end with 95% organic phase. Pay attention to washing with a large proportion of organic phase at the end of the gradient for a few minutes according to the actual situation to ensure the The impurities of small polarity are eluted to prevent the sample from remaining in the next needle. The slope of the gradient generally adopts a concave-line type that is small first and then large, and the gradient changes first slowly and then fast, and then optimize the gradient on this basis.
The initial ratio of the gradient aqueous phase of the buffer salt solution should generally start from 10 to 20%. In order to prevent salt precipitation, avoid washing with pure organic phase at the end of the gradient, and use a constant gradient slope. Based on this Adjust the gradient as the method runs.
Generally, the sample collection time of an API is controlled at about 40-50 minutes, and the peak of the sample is preferably about 15-20 minutes. If there are impurities with very small polarity, a large proportion of organic solvents can be added for a period of time to wash the chromatogram at the end. Column, and finally set a re-equilibration time of about 10 minutes. Intermediate and IPC sample analysis method time can be halved or shorter as needed.
Analytical method development requires diode array detector, chromatographic peak purity check and selection of detection wavelength, through chromatographic peak purity check to ensure that there are no other impurities in the main peak, the requirement for wavelength selection for purity detection is relatively simple, and the principle is to use as many as possible The impurities are reflected in the chromatogram. Many impurities have UV absorption only at low wavelengths, so we choose the lowest possible wavelength. The cut-off wavelength of acetonitrile is 190~195nm, and the detection wavelength of acetonitrile can be selected at 210~220nm. Some companies also require that the strongest UV absorption band of the product be selected for comparison with the two bands of 210~220nm, whichever is lower in purity, so that the quality of the product can be more strictly controlled.
In order to ensure the accuracy and reliability of the analysis and testing results, the accuracy, scientificity and feasibility of the analysis methods used must be verified to prove that the analysis methods meet the purpose and requirements of the testing. This is the analysis method verification. Essentially, method verification is to set up certain verification content in advance according to the requirements of the test item, and verify that the used analysis method meets the requirements of the test item through a reasonable test. Method validation has an important role and significance in quality control. Only validated analytical methods can be used in the analysis and testing of pharmaceutical production. Method validation is the basis for formulating quality standards. Method verification content includes method specificity, linearity, range, accuracy, precision, detection limit, quantification limit, durability and system applicability, etc. The verification requirements are different for different testing purposes.
Specificity refers to the characteristic that the analytical method can separate the product from the impurities, also known as selectivity. For purity testing, the known impurities in the product can be added to the standard product, or the crude product can be used directly to investigate whether the product peak is interfered by impurities. For process tracking, reaction system samples can be used to investigate whether there are other impurities interfered. If necessary, use a diode array detector or a mass spectrometer to check the purity of the chromatographic peak. Generally, the separation degree between product and impurities is greater than 2.0.
Linearity is the degree to which the test result has a linear relationship with the concentration of the raw material or product in the sample within the set range. Linearity is the basis of quantitative testing, and all items that require quantitative testing need to verify linearity. Generally, the stock solution is accurately diluted, or the samples are accurately weighed separately, to prepare a series of concentrations of the tested substances (more than 5), run the sequence from small to large concentration, and plot the peak area and concentration as a function of the minimum two Multiplication is used for linear regression calculation, and the linearity of the analysis method is investigated.
Range refers to the concentration interval of the analyte in the sample when a certain degree of accuracy, precision and linearity can be achieved. Simply put, the range is the maximum and minimum concentration of the analyte in the sample to which the analytical method is applicable. All analytical methods that require quantitative detection need to verify the range. For purity detection, the range should be 80% to 120% of the test concentration.
Accuracy refers to the degree of closeness between the measured result and the true value, so it is also called authenticity. It is necessary to verify the accuracy of quantitative analysis methods. The accuracy should be established within the specified range. For APIs, standard products of known purity or APIs that meet the requirements can be used for determination. If necessary, the results can be compared with another method with established accuracy.
Precision refers to the degree of closeness between the results of a series of tests on the same uniform sample after multiple samplings under specified conditions. Precision is generally expressed by relative standard deviation, and the number of sampling tests should be at least 6 times.
Precision can be investigated from three levels: repeatability, intermediate precision, and reproducibility.
a. Repeatability is the precision of the results measured by the same analyst in a short time interval under the same operating conditions. It is generally evaluated by measuring the results of 6 times with a sample with a concentration level of 100%.
b. Intermediate precision: The precision of the measurement results in the same laboratory when the internal conditions such as the date, analysts, and instruments change.
c. Reproducibility: refers to the precision of the measurement results of different analysts in different laboratories.
The detection limit means that the analyte in the sample can be detected the minimum amount reached does not need to be accurately quantified. The detection limit reflects the sensitivity of the analytical method. The detection limit can be determined by testing a series of samples with known concentrations of the analyte to determine the minimum concentration of the analyte that can be accurately and reliably detected. It can also be used to determine the signal and noise signal of the sample with a known concentration. For comparison, the detection limit is determined by the concentration when the signal-to-noise ratio is 3:1, and it is generally required to reach 0.05% of the injection concentration.
The limit of quantification refers to the minimum amount of the analyte in the sample that can be quantitatively detected. The determination result requires a certain degree of accuracy and precision. The limit of quantification reflects the sensitive and quantitative detection ability of the analytical method. The detection requires strict control of the content of impurities, and the quantification limit of the method must be investigated to ensure that the impurities can be accurately quantified. Generally, the quantification limit is determined by the corresponding concentration or injection volume when the signal-to-noise ratio is 10:1.
Durability refers to the degree to which the measurement results are not affected when the measurement conditions undergo small changes. Durability mainly indicates the anti-interference ability of the method. The main variable factors include: mobile phase composition, flow rate and pH value, chromatographic column, Column temperature and so on. After testing, it should be explained whether small changes can meet the requirements of the system suitability test to ensure that the method is effective.
Liquid chromatography analysis methods mainly rely on high-performance liquid chromatographs and chromatographic columns. When performing method verification, it is necessary to use high-performance liquid chromatographs, chromatographic columns, mobile phases as well as experimental operations and samples to be tested as a complete system. Carry out the evaluation, and take the system applicability as an integral part of the analysis method. The system applicability is the index for evaluating the entire system. The general system adaptability requirements are: the analytical method can reach the detection limit of 0.05%, the tailing factor of the main peak is 0.5<Tf<2.5, the separation between the main peak and the impurities is greater than 2.0, the blank is clean, and there is no system peak interference at the main peak.
Analysis method development and verification are a whole. In actual work, the analysis method is generally developed first, and method verification is done after proper optimization. Part of the verification content is to be done when the analysis method is developed, such as the exclusive analysis method. Sexual verification. Analytical method verification does not have to verify all the content, as long as the verification content is sufficient to prove the rationality of the analytical method, such as impurity limit detection generally only needs to verify the specificity and detection limit, while the precision, linearity and quantification limit For items that involve quantitative measurement, verification is not required.
Sometimes the analytical method needs to be fully or partially revalidated. When the API synthesis process is changed, new impurities may be introduced. The specificity of the impurity inspection method and the content determination method needs to be verified again to prove that the related substance inspection method can detect the newly introduced impurities and the newly introduced impurities The content determination of the main component should be without interference. When the analysis method is partially changed, if the detection wavelength changes, it is necessary to re-verify the detection limit, specificity, accuracy, precision, linearity, etc., to prove the rationality and feasibility of the changed analysis method.
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