CN103439440A - Prediction method for retention time of high performance liquid chromatographic peak - Google Patents
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Abstract
The invention relates to a prediction method for retention time of a high performance liquid chromatographic peak. The method comprises the following steps: measuring standard retention time of a variety of components of a variety of samples; choosing two components from target components of each of the samples as dual standard control components of the sample; measuring actual retention time of the dual standard control components in a test solution of a to-be-measured sample; measuring actual retention time of other target components; carrying out two-point verification and multi-point verification; etc. With the prediction method, retention time of chromatographic peaks of a variety of components in the to-be-measured sample can be accurately predicted, then nature determination is carried out on the chromatographic peaks of the to-be-measured sample, and discrimination of the to-be-measured sample is carried out. The method provided by the invention has high prediction precision, is applicable to more chromatographic columns and is obviously superior to relative retention time methods in the prior art.
Description
Technical field
The present invention relates to a kind of high performance liquid chromatography peak retention time prediction method, belong to high performance liquid chromatography detection technique field.
Background technology
The assay of Chinese medicine multi-target ingredient, fingerprint map analyzing, in the work such as related substance inspection of chemical drugs (or microbiotic), core missions are exactly that target compound to One's name is legion carries out qualitative, if all use pure chemical reference substance, to bring great difficulty to preparation and the supply of standard substance, and also can significantly improve testing cost.Therefore qualitative by coming someway other chromatographic peaks are carried out with a small amount of chemical reference substance, be of great significance and value.
At present, the relative retention time method of using a chemical reference substance is the most frequently used chromatographic peak qualitative method, is widely used in the related substance inspection in America and Europe and Chinese Pharmacopoeia, also many uses in many indexs assay research of alternating pattern.But, due to the difference of chromatograph and chromatographic column, predicted value and the deviation between measured value of retention time are usually larger, need to limit the model of chromatographic column in practical application, or select chromatographic column of the same type, that separating property is similar, and inconvenience is arranged more.
Summary of the invention
For solving the problems of the technologies described above, the object of the present invention is to provide a kind of high performance liquid chromatography peak retention time prediction method, carry out chromatographic peak by two chemical reference substances qualitative, than the relative retention time method, the method can significantly reduce error qualitatively, increases the chromatographic column quantity of using.
For achieving the above object, the invention provides a kind of high performance liquid chromatography (HPLC) peak retention time prediction method, can be called two graticule correction methods, it comprises the following steps:
(1) utilize high performance liquid chromatograph to measure the actual measurement retention time of various compositions on many different chromatographic columns of various samples, actual measurement retention time to the various compositions of the same sample measured on different chromatographic columns is carried out two groups by two groups linear fit, drawing standard curve calculate correlation coefficient r respectively, reject the actual measurement retention time of (chromatographic column is measured) r<0.995, (chromatographic column is measured) departs from the actual measurement retention time that larger actual measurement retention time and (chromatographic column is measured) cause chromatographic peak sequentially to change, the mean value of the actual measurement retention time that the chromatographic column be left of usining is measured is as the standard retention time of various compositions,
Perhaps, the mean value of actual measurement retention time of same composition of the same sample measured on all chromatographic columns of take is first plan standard retention time, actual measurement retention time with all the components of the same sample measured on the first plan standard retention time of all the components and all chromatographic columns is carried out respectively linear fit, drawing standard curve calculate correlation coefficient r respectively, reject the actual measurement retention time of (chromatographic column is measured) r<0.995, (chromatographic column is measured) departs from the actual measurement retention time that larger actual measurement retention time and (chromatographic column is measured) cause chromatographic peak sequentially to change, the mean value of the actual measurement retention time that the chromatographic column be left of usining is measured is as the standard retention time of various compositions,
(2) select the two mark contrast compositions of two compositions as this sample in the target component of each sample;
(3) on a root chromatogram column, the need testing solution of operation testing sample and two mark contrast the reference substance solution of compositions and utilize high performance liquid chromatograph to be detected, obtain the chromatogram of need testing solution, reference substance by two marks contrast compositions carries out qualitative to the chromatographic peak in obtained chromatogram, obtain the actual measurement retention times of two mark contrast compositions in need testing solution;
(4) take the standard retention time of two marks contrast compositions sets up linearity curve as ordinate, actual measurement retention time as horizontal ordinate, and obtains two point Linear equations;
(5) bring the standard retention time of the target component of the testing sample except two mark contrast compositions into described two point Linear equations, obtain the prediction retention time of respective objects composition, according to this prediction retention time, other chromatographic peaks in the chromatogram of test sample are carried out qualitatively, obtain the actual measurement retention time of respective objects composition;
If the absolute value of the standard retention time of the target component of all testing samples except two mark contrast compositions and the deviation of actual measurement retention time all>t
2, prediction of failure, the chromatographic column in exchonge step (3), and repeating step (3)-(5);
If have the absolute value≤t of standard retention time with the deviation of actual measurement retention time of a target component among the target component of the testing sample except two mark contrast compositions at least
2, enter step (6);
(6) absolute value≤t with the deviation of prediction retention time with all actual measurement retention times
2the target component standard retention times that contrasts composition with two marks be ordinate, to survey retention time be that horizontal ordinate is set up the multiple spot relation curve and obtained the linear multi equation;
(7) bring the standard retention time of the target component of the testing sample except two mark contrast compositions into the linear multi equation, calculate the new prediction retention time of respective objects composition;
(8) in the chromatogram of test sample, find and the new immediate chromatographic peak of prediction retention time, obtain the new actual measurement retention time of respective objects composition, and calculate the absolute value of the deviation of the new actual measurement retention time of each target component and new prediction retention time, whether comprised all target components to judge the linear multi equation: if the absolute value≤t of all deviations
2, enter next step, if absolute value>t devious
2, return to step (6), with the absolute value≤t devious calculated in step (8)
2the target component standard retention times that contrasts composition with two marks be ordinate, to survey retention time be that horizontal ordinate re-establishes the multiple spot relation curve and obtains new linear multi equation, repeating step (6)-(8);
(9) detect the standard retention time of all target components and new prediction retention time deviation absolute value whether≤t
m, t wherein
m<t
2: if predict successfully; If not, prediction of failure; During prediction of failure, the chromatographic column in exchonge step (3), repeating step (3)-(9).
The purpose of said method provided by the present invention is the retention time of the chromatographic peak of the various compositions in order to obtain testing sample, and carry out qualitative to chromatographic peak, utilize two chemical reference substances to reach qualitative to a plurality of target components in testing sample, applicable sample can be Chinese medicine, natural plant, chemical drugs, food (such as health food) or microbiotic etc., but be not limited to this, anyly need to carry out multicomponent by HPLC and all can be suitable in field qualitatively.
In said method, preferably, during actual measurement retention time at the various compositions that utilize high performance liquid chromatograph to measure various samples on many root chromatogram columns, at the equal sample introduction 3 times at least of each root chromatogram column, and the RSD of resulting actual measurement retention time should≤2%.
In said method, preferably, the standard retention time of selected two mark reference substance compositions is near the two ends of the retention time section of the target component of corresponding sample.The retention time section here refers to the retention time scope of the chromatographic peak of various compositions.
In said method, preferably, the retention time section of the target component of sample is divided into to four sections, the standard retention times of selected two mark reference substance compositions are positioned near first separation and the 3rd separation.
In said method, preferably, while selecting the standard retention time of two mark reference substance compositions, be not chosen in the chromatographic column fit procedure and depart from larger composition, and preferred composition cheap and easy to get.
In said method, preferably, t
2<1min, t
m<1min; More preferably, t
2for 0.7min, t
mfor 0.5min.
In said method, adopt more chromatographic columns to be measured, can obtain more data, and the data that add are more, error is just less, but a decreasing returns effect is also arranged, preferably, in step (1), the actual measurement retention time that the 10-20 root chromatogram column adopted during the standard retention time of various compositions is measured is calculated, thus obtained standard retention time has enough representativenesses, should select more chromatographic column when selecting chromatographic column to be surveyed retention time mensuration at the beginning, this requirement of the data that adopt the 10-20 root chromatogram column to record here while calculating to meet.
According to the specific embodiment of the invention scheme, above-mentioned high performance liquid chromatography peak retention time prediction method can carry out in such a way, and its idiographic flow can be as shown in Figure 1:
1, chromatographic column matching
(1) utilize high performance liquid chromatograph to measure the actual measurement retention time (at least retain 3 decimals) of various compositions on many different chromatographic columns of various samples, this detection can adopt chromatographic condition (kind, the ratio that comprise column temperature, flow velocity and mobile phase of optimization, can official record be selected) carry out, at least want sample introduction three times in testing process, and meet the RSD of retention time≤2%, to control the error of image data;
Actual measurement retention time to the various compositions of the same sample measured on different chromatographic columns is carried out two groups by two groups linear fit, drawing standard curve calculate correlation coefficient r respectively, rejecting departs from the chromatographic column that larger chromatographic column even causes chromatographic peak sequentially to change, in general, the fitting effect of these chromatographic columns and other chromatographic columns is not good, shows as r<0.995 or certain (or some) point and obviously departs from the straight line of matching; Rejecting these data can make the data such as standard retention time of acquisition more accurate; When rejecting, can be classified to chromatographic column, will matching is reasonable each other put together, then selected, select a fairly large number of one group as far as possible;
After rejecting, the mean value of the actual measurement retention time that the not disallowable chromatographic column of usining is measured is as standard retention time (the standard retention time of various compositions, be called for short SRT), using this standard retention time as the reference value of predicting, the data that should adopt the 10-20 root chromatogram column to measure during calculating are carried out.
(2) select the two mark contrast compositions of two compositions as this sample in the target component of each sample; In theory, any two compositions in object should be all the same as two target effects, but in fact due to difference, the complexity of elution requirement and the accidental error of stratographic analysis of structure between chromatographic column and chromatographic difference, composition, two targets are selected or can be caused difference, in addition, also to take into account complexity and the price factor that this reference substance obtains.Be below that two marks are selected the principle that follow and take into account: two marks should be distributed in the two ends of target compound retention time section as far as possible, and optimal location is after the retention time section is divided into to 4 sections, near first and the 3 two separation; Two mark compositions are tried not to be chosen in the chromatographic column fit procedure and are departed from larger composition; Two mark compositions are selected composition cheap and easy to get as far as possible.
After obtaining two mark contrast compositions of the SRT of various compositions of various samples and various samples, it can be summarised in together as regular set or pharmacopeia, in the time need to being predicted the retention time of the chromatographic peak of a certain sample, can from this regular set or pharmacopeia, inquire about its corresponding SRT and two mark contrast composition, then be predicted according to following steps:
2, two point predictions:
(3) at a root chromatogram column, (can be chromatographic column arbitrarily, the chromatographic column adopted while not necessarily setting up above-mentioned regular set or pharmacopeia) need testing solution of upper operation testing sample is marked the reference substance solution of contrast compositions and utilizes high performance liquid chromatograph to be detected with two, obtain the chromatogram of need testing solution, reference substance by two marks contrast compositions carries out qualitative to the chromatographic peak in obtained chromatogram, obtain the actual measurement retention times of two mark contrast compositions in need testing solution;
(4) take the standard retention time of two marks contrast compositions sets up linearity curve as ordinate, actual measurement retention time as horizontal ordinate, and obtains two point Linear equations;
(5) bring the standard retention time of the target component of the testing sample except two mark contrast compositions into described two point Linear equations, obtain the prediction retention time of respective objects composition, according to this prediction retention time, other chromatographic peaks in the chromatogram of test sample are carried out qualitatively, obtain the actual measurement retention time of respective objects composition;
If except two mark contrast compositions, the absolute value of the deviation of the standard retention time of the target component that testing sample is all and actual measurement retention time all>t
2, prediction of failure, the chromatographic column in exchonge step (3), and repeating step (3)-(5);
If except two mark contrast compositions, has the absolute value≤t of standard retention time with the deviation of actual measurement retention time of a target component among the target component of testing sample at least
2, enter step (6).
3, multiple spot prediction:
(6) absolute value≤t with the deviation of prediction retention time with all actual measurement retention times
2the target component standard retention times that contrasts composition with two marks be ordinate, to survey retention time be that horizontal ordinate is set up the multiple spot relation curve and obtained the linear multi equation;
(7) bring the standard retention time of the target component of the testing sample except two mark contrast compositions into the linear multi equation, calculate the new prediction retention time of respective objects composition;
(8) in the chromatogram of test sample, find and the new immediate chromatographic peak of prediction retention time, obtain the new actual measurement retention time of respective objects composition, and calculate the absolute value of the deviation of the new actual measurement retention time of each target component and new prediction retention time, whether comprised all target components to judge the linear multi equation: if the absolute value≤t of all deviations
2, enter next step, if absolute value>t devious
2, return to step (6), even only have an absolute value>t
2also to return to step (6), with the absolute value≤t devious calculated in step (8)
2the target component standard retention times that contrasts composition with two marks be ordinate, to survey retention time be that horizontal ordinate re-establishes the multiple spot relation curve and obtains new linear multi equation, repeating step (6)-(8), until the linear multi equation of setting up can comprise all points;
When step (6) is set up the linear multi equation at the beginning, may have the data of target component partly because absolute value>the t of deviation
2and do not use, when in step (7), carrying it into the linear multi equation, likely can the become≤t of absolute value of the new prediction retention time calculated and the difference of standard retention time
2, therefore, as absolute value>t devious
2and return to step (6) afterwards, and what the data that adopt while setting up new linear multi equation can be from before is different, and follow-up process also has difference, when all data can both meet the absolute value≤t of deviation
2afterwards, just can enter follow-up step (9).
(9) detect the standard retention time of all target components and new prediction retention time deviation absolute value whether≤t
m, and t
m<t
2: if predict successfully; If not, prediction of failure; During prediction of failure, the chromatographic column in exchonge step (3), repeating step (3)-(9).
Multiple spot prediction by back can improve the precision of prediction.In addition, t
2>t
mcan reach the effect of wide-in and strict-out, improve on the one hand the quantity of applicable post, also guarantee on the other hand the accuracy of prediction, as for above-mentioned t
2, t
mconcrete numerical value, can according to circumstances be adjusted, generally be not more than 1min.
Adopt the retention time of the chromatographic peak of the various compositions that high performance liquid chromatography provided by the invention peak retention time prediction method can the Accurate Prediction testing sample, and then the chromatographic peak of testing sample is carried out qualitative, carry out the discriminating of testing sample.Method provided by the present invention has higher precision of prediction, and applicable chromatographic column quantity is many, obviously is better than existing relative retention time method.
The accompanying drawing explanation
The process flow diagram that Fig. 1 is high performance liquid chromatography provided by the invention peak retention time prediction method.
The fitting a straight line figure that Fig. 2 is two root chromatogram columns.
Fig. 3 a-Fig. 3 c is the fitting result of 5 kinds of anthraquinones on 10 root chromatogram columns in rheum officinale.
The linear relationship chart of the retention time that Fig. 4 a-Fig. 4 c is five kinds of compositions of rheum officinale of measuring on different chromatographic columns.
The chromatogram that Fig. 5 is need testing solution.
Embodiment
Understand for technical characterictic of the present invention, purpose and beneficial effect being had more clearly, now technical scheme of the present invention is carried out to following detailed description, but but can not be interpreted as the restriction to practical range of the present invention.
Embodiment
The present embodiment provides a kind of high performance liquid chromatography peak retention time prediction method, and it carries out the prediction of chromatographic peak retention time to rheum officinale, comprise the following steps:
1, chromatographic column matching:
According to the chromatographic condition of optimizing (according to " the assay item (the 22nd page) of a rheum officinale of Chinese pharmacopoeia version in 2010 is determined, design parameter is that mobile phase is methyl alcohol-0.1% phosphate aqueous solution (85:15), detect wavelength 254nm, 30 ℃ of column temperatures, flow velocity 1.0mLmin
-1), at the C18 of the different brands of 11, model column sample injection mixing reference substance or need testing solution, obtain the actual measurement retention time of five kinds of compositions (aloe-emodin, Rhein, archen, Chrysophanol, Physcion) on different chromatographic columns, at least sample introduction three times and will retain three decimals, remove the chromatographic column that RSD is greater than 2%, the chromatographic column adopted has 12, be respectively: Kromasil C18*, Sunfire C18**, Agilent TC-C18*, Inertsil C18**, Hyperisil C18*, Vensil MP C18**, Shimadzu VP C18**, Phenomen luna C18**, Agilent HC-C18*, Agilent Zorbax SB-C18*, Shiseido AQ C18*, Shiseido MG C18*, above C18 post is referred to as C1 to C12(*.4.6mm * 250mm * 5 μ m, * .4.6mm * 150mm * 5 μ m), wherein C1 to C11 is for the chromatographic column matching, C12 is used for method validation (having neither part nor lot in the calculating of SRT) as unknown post,
The actual measurement retention time of above-mentioned five kinds of compositions that C1 to C11 chromatographic column is measured is carried out linear fit (combination of two), the drawing standard curve, calculate correlation coefficient r, then reject and depart from the chromatographic column that even causes chromatographic peak sequentially to change greatly (for example the chromatographic column of r<0.995, and indivedual point obviously departs from the chromatographic column of fitting a straight line); For example, shown in Fig. 2, its fitting a straight line that is C2, C11 two root chromatogram columns, can find out, second point (Rhein) of C11 chromatographic column located to depart from greatlyr with the C2 chromatographic column, and this two root chromatogram column can not be put together, should reject one; By matching, retained C1 to C10, C11 rejects.
2, determining of SRT:
Adopt " the content assaying method under Chinese pharmacopoeia version rheum officinale item in 2010 on 2 chromatographic systems, measure aloe-emodin, Rhein, archen, Chrysophanol and 5 kinds of dissociated anthraquinone compositions of Physcion (above 5 kinds of compositions sort from small to large by retention time) wherein, the corresponding post 1,2 of system 1: Shimadzu 2010A() simultaneously; The corresponding post 3,4 of system 2:waters e2695() and the chromatographic column of 4 different brands and model, post 1:kromasil C18 (250mm * 4.6mm * 5 μ m); Post 2:waters sunfire C18 (150mm * 4.6mm * 5 μ m); Post 3:agilent TC-C18 (250mm * 4.6mm * 5 μ m); Post 4:inertsil C18 (150mm * 4.6mm * 5 μ m), with accuracy and the applicability of verifying this conclusion, the results are shown in Figure 4a-Fig. 4 c, and wherein, horizontal, ordinate is respectively the retention time of 5 kinds of compositions on any two root chromatogram columns.
Fig. 3 a-Fig. 3 c is the fitting results of 5 kinds of anthraquinones of rheum officinale on 10 root chromatogram columns, the SRT that horizontal ordinate is 5 kinds of anthraquinones, and ordinate is the actual measurement retention time on each chromatographic column, the linear equation of each chromatographic column and related coefficient are in Table 1.
Linear equation and the related coefficient of retention time on the different chromatographic columns of table 1
| Chromatographic column | Linear equation | Related coefficient |
| C1 | Y=1.7985x-2.5911 | 0.9987 |
| C2 | Y=0.7319x+0.1108 | 0.9998 |
| C3 | Y=0.6900x+2.4103 | 0.9962 |
| C4 | Y=1.0901x-0.9971 | 0.9985 |
| C5 | Y=1.0790x-0.0185 | 0.9987 |
| C6 | Y=1.0438x-0.0284 | 0.9963 |
| C7 | Y=0.9682x-0.3483 | 0.9998 |
| C8 | Y=0.5612x+0.7563 | 0.9982 |
| C9 | Y=1.1173x+0.4010 | 0.9999 |
| C10 | Y=0.8448x+0.6422 | 0.9978 |
Comparison diagram 3a-Fig. 3 c and Fig. 4 a-Fig. 4 c can find out, the r of C2 is from 0.9976(r
2=0.9951) brought up to the r of 0.9998, C4 from 0.9974(r
2=0.9947) brought up to the r of 0.9985, C3 from 0.9977(r
2=0.9954) be reduced to 0.9962, belonged to small probability event, from whole data, use SRT's is with the obvious advantage.
The mean value of the actual measurement retention time that not disallowable above-mentioned 10 root chromatogram columns of usining are measured is as the SRT of various compositions, using this SRT as the reference value of predicting, the SRT of five kinds of compositions is respectively: aloe-emodin 4.081min, Rhein 4.979min, archen 7.380min, Chrysophanol 9.385min and Physcion 12.715min, two marks are selected the shortest aloe-emodin and the longest Physcion of retention time.
3, two point predictions
At first, at a C18 post (C12, Shiseido MG C18*) upper operation two mark reference substance solution (being aloe-emodin reference substance solution and Physcion reference substance) and need testing solution (being the solution of testing sample), by reference substance, to the chromatographic peak in test sample chromatogram (as shown in Figure 5), carry out qualitative, thereby obtain the actual measurement retention time of two mark compositions in test sample: aloe-emodin 4.970min, Physcion 16.000min;
Then, take SRT as ordinate, actual measurement retention time are horizontal ordinate, obtain 2 points: aloe-emodin (4.970,4.081) and Physcion (16.000,12.715), and then obtain two point Linear equation: Y=0.7828X+0.1903;
By the above-mentioned equation of the SRT value of all the other 3 kinds of anthraquinones (corresponding Y value) substitution, obtain predicting retention time: Rhein 6.117min, archen 9.185min, Chrysophanol 11.746min;
Finally, in the test sample chromatogram, with the immediate chromatographic peak of predicted value, (a is the reference substance solution chromatogram in searching, b is rheum officinale need testing solution chromatogram, 1,5 is two marks, 2,3,4 is target peak), and obtain surveying retention time, be followed successively by: Rhein 5.978min, archen 9.218min and Chrysophanol 11.843min, the absolute value of the deviation of actual measurement retention time and prediction retention time is followed successively by 0.139min, 0.033min and 0.097min.
4, multiple spot prediction:
(1) deviation is judged: the absolute value of the deviation of Rhein obtained above, archen, Chrysophanol all is less than 0.7min, and therefore, prediction effect is good, and these three kinds of objects all participate in matching linear multi equation;
(2) matching linear multi equation: take SRT as ordinate, the actual measurement retention time is horizontal ordinate, obtain 5 points: aloe-emodin (4.970,4.081), Rhein (5.978,4.979), archen (9.218,7.380), Chrysophanol (11.843,9.385) and Physcion (16.000,12.715), and then obtain 5 point Linear equation: Y=0.7759X+0.2580;
(3) by the SRT value of all the other 3 kinds of anthraquinones (Rhein, archen, Chrysophanol) substitution equation again, obtain new prediction retention time: Rhein 6.085min, archen 9.179min, Chrysophanol 11.763min;
(4) again find the new immediate chromatographic peak of prediction retention time with 3 kinds of anthraquinones (Rhein, archen, Chrysophanol) in the chromatogram of test sample, be still 2,3, No. 4 peaks, and obtain the new actual measurement retention time of 3 kinds of anthraquinones (Rhein, archen, Chrysophanol): Rhein 5.978min, archen 9.218min and Chrysophanol 11.843min;
(5) whether the matching of inspection linear multi equation has comprised all objects, the absolute value that checks whether all object prediction deviations all≤0.5min, the prediction deviation absolute value of 3 kinds of anthraquinones (Rhein, archen, Chrysophanol) is followed successively by 0.107min, 0.033min and 0.080min, all be less than 0.5min, therefore, predict successfully.
The comparison of two graticule correction methods and relative retention time method prediction effect
The 5 kinds of anthraquinone analyses in rheum officinale of take are the relatively qualities of two kinds of Forecasting Methodologies of example.Table 2,3 is that (prediction of relative retention time method be take archen as object of reference to the comparative result of two methods on 10 root chromatogram columns, the average of relative retention time is 0.556,0.678,1.266 and 1.709), wherein, in table 3, secondary series is prediction retention time and the maximum absolute deviation of surveying retention time; The 3rd row are corresponding maximum relative deviations; The 4th row are that the number that absolute deviation is less than or equal to 0.5min accounts for total number percent; The 5th classifies the sum (when the absolute deviation of each ingredient prediction value all is less than or equal to 0.5min on certain chromatographic column, thinking that this post is applicable) of applicable post as; The 6th row and the 7th are classified number percent and the applicable post number of corresponding relative deviation as.
Absolute deviation/the min of table 2 predicting retention time value
The comparison that predicts the outcome of two kinds of methods of table 3
By the result shown in table 2 and table 3, can be found out, the precision of prediction of two graticule correction methods is higher, and applicable chromatographic column is more, obviously is better than the relative retention time method.
Claims (9)
1. a high performance liquid chromatography peak retention time prediction method, it comprises the following steps:
(1) utilize high performance liquid chromatograph to measure the actual measurement retention time of various compositions on many different chromatographic columns of various samples, actual measurement retention time to the various compositions of the same sample measured on different chromatographic columns is carried out two groups by two groups linear fit, drawing standard curve calculate correlation coefficient r respectively, reject the actual measurement retention time of the chromatographic column mensuration of r<0.995, the actual measurement retention time that the chromatographic column that departs from the actual measurement retention time of larger chromatographic column mensuration and cause chromatographic peak sequentially to change is measured, the mean value of the actual measurement retention time that the chromatographic column be left of usining is measured is as the standard retention time of various compositions,
Perhaps, the mean value of actual measurement retention time of same composition of the same sample measured on all chromatographic columns of take is first plan standard retention time, actual measurement retention time with all the components of the same sample measured on the first plan standard retention time of all the components and all chromatographic columns is carried out respectively linear fit, drawing standard curve calculate correlation coefficient r respectively, reject the actual measurement retention time of the chromatographic column mensuration of r<0.995, the actual measurement retention time that the chromatographic column that departs from the actual measurement retention time of larger chromatographic column mensuration and cause chromatographic peak sequentially to change is measured, the mean value of the actual measurement retention time that the chromatographic column be left of usining is measured is as the standard retention time of various compositions,
(2) select the two mark contrast compositions of two compositions as this sample in the target component of each sample;
(3) on a root chromatogram column, the need testing solution of operation testing sample and two mark contrast the reference substance solution of compositions and utilize high performance liquid chromatograph to be detected, obtain the chromatogram of need testing solution, reference substance by two marks contrast compositions carries out qualitative to the chromatographic peak in obtained chromatogram, obtain the actual measurement retention times of two mark contrast compositions in need testing solution;
(4) take the standard retention time of two marks contrast compositions sets up linearity curve as ordinate, actual measurement retention time as horizontal ordinate, and obtains two point Linear equations;
(5) bring the standard retention time of the target component of the testing sample except two mark contrast compositions into described two point Linear equations, obtain the prediction retention time of respective objects composition, according to this prediction retention time, other chromatographic peaks in the chromatogram of test sample are carried out qualitatively, obtain the actual measurement retention time of respective objects composition;
If the absolute value of the standard retention time of the target component of all testing samples except two mark contrast compositions and the deviation of actual measurement retention time all>t
2, prediction of failure, the chromatographic column in exchonge step (3), and repeating step (3)-(5);
If have the absolute value≤t of standard retention time with the deviation of actual measurement retention time of a target component among the target component of the testing sample except two mark contrast compositions at least
2, enter step (6);
(6) absolute value≤t with the deviation of prediction retention time with all actual measurement retention times
2the target component standard retention times that contrasts composition with two marks be ordinate, to survey retention time be that horizontal ordinate is set up the multiple spot relation curve and obtained the linear multi equation;
(7) bring the standard retention time of the target component of the testing sample except two mark contrast compositions into the linear multi equation, calculate the new prediction retention time of respective objects composition;
(8) in the chromatogram of test sample, find and the new immediate chromatographic peak of prediction retention time, obtain the new actual measurement retention time of respective objects composition, and calculate the absolute value of the deviation of the new actual measurement retention time of each target component and new prediction retention time, whether comprised all target components to judge the linear multi equation: if the absolute value≤t of all deviations
2, enter next step, if absolute value>t devious
2, return to step (6), with the absolute value≤t devious calculated in step (8)
2the target component standard retention times that contrasts composition with two marks be ordinate, to survey retention time be that horizontal ordinate re-establishes the multiple spot relation curve and obtains new linear multi equation, repeating step (6)-(8);
(9) detect the standard retention time of all target components and new prediction retention time deviation absolute value whether≤t
m, and t
m<t
2: if predict successfully; If not, prediction of failure; During prediction of failure, the chromatographic column in exchonge step (3), repeating step (3)-(9).
2. high performance liquid chromatography according to claim 1 peak retention time prediction method, wherein, described sample is Chinese medicine, natural plant, chemical drugs, food or microbiotic.
3. high performance liquid chromatography according to claim 1 peak retention time prediction method, wherein, during actual measurement retention time at the various compositions that utilize high performance liquid chromatograph to measure various samples on many root chromatogram columns, at the equal sample introduction 3 times at least of each root chromatogram column, and the RSD of resulting actual measurement retention time≤2%.
4. high performance liquid chromatography according to claim 1 peak retention time prediction method, wherein, mark the two ends of the standard retention time of reference substance compositions near the retention time section of the target component of sample for selected pair.
5. according to the described high performance liquid chromatography of claim 1 or 4 peak retention time prediction method, wherein, the retention time section of the target component of sample is divided into to four sections, and the standard retention times of selected two mark reference substance compositions are positioned near first separation and the 3rd separation.
6. according to the described high performance liquid chromatography of claim 4 or 5 peak retention time prediction method, wherein, while selecting the standard retention time of two mark reference substance compositions, be not chosen in the chromatographic column fit procedure and depart from larger composition, preferably composition cheap and easy to get.
7. high performance liquid chromatography according to claim 1 peak retention time prediction method, wherein, described t
2<1min, t
m<1min.
8. high performance liquid chromatography according to claim 7 peak retention time prediction method, wherein, described t
2for 0.7min, described t
mfor 0.5min.
9. high performance liquid chromatography according to claim 1 peak retention time prediction method, wherein, in step (1), the actual measurement retention time that the 10-20 root chromatogram column adopted during the standard retention time of various compositions is measured is calculated.
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