ISSN 1061-9348, Journal of Analytical Chemistry, 2016, Vol. 71, No. 8, pp. 794–802. © Pleiades Publishing, Ltd., 2016.
ARTICLES
Simple Approach for Evaluation of Matrix Effect in the Mass Spectrometry of Synthetic Cannabinoids1 P. Adamowicz* and W. Wrzesień Institute of Forensic Research, Westerplatte 9, 31-033 Krakow, Poland *e-mail:
[email protected] Received October 28, 2015; in final form, January 19, 2016
Abstract—A simple approach for studying and identifying matrix effect is described. This method for the determination of matrix effect combines the advantages of two most popular traditional methods while eliminating their disadvantages. A postcolumn infusion system was used to observe the MS signal alterations of synthetic cannabinoids: UR-144, XLR-11 and STS-135. Protein precipitation, liquid–liquid extraction and solid phase extraction sample preparation methods were tested. The results of the experiments showed that the discussed method of matrix effect estimation can have practical application in the development of analytical methods. The comparison of the normalized matrix effect profiles can be done even for data obtained over time. Obtained results also indicated that matrix effect was highly dependent on sample preparation. Although similar structure, significant differences were observed for different synthetic cannabinoids. Keywords: matrix effect, LC–MS, sample preparation, synthetic cannabinoids DOI: 10.1134/S1061934816080025
Liquid chromatography coupled with mass spectrometry (LC–MS) is an analytical technique which combines the possibility of separating the components of a sample by liquid chromatography with the possibility of their identification using the mass spectrometry detection. LC–MS is the current analytical method of choice for quantitation of analytes in biological samples. This technique is used in many areas of chemistry including pharmacokinetic studies, analysis of pharmaceuticals, clinical and forensic toxicology and proteomics, and is therefore the most commonly used technique in the field of bioanalyses. The advantage of the mass detector is that it is very sensitive and specific in comparison to the other detectors. Despite the huge prevalence, this technique has some limitations. Components of the matrix can affect the most important parameters of methods that are linearity, precision and the accuracy of determinations, which can lead to erroneous quantification of analyte. These influences are known as matrix effects and are mostly unseen in the chromatogram. These effects can be observed in the analysis of many substances, and are particularly noticeable when analyzing an extremely complex matrix like blood or urine. Low purity biological samples can cause elevated background and other negative effects. Matrix effects 1 The article is published in the original.
can occur when the analyte is eluted together with other compounds, both endogenous (phospholipids in particular) and exogenous (drugs and/or metabolites, internal standards, and additives to the mobile phase). Generally matrix effect is caused by all other components of the sample except the specific compound to be quantified. Coeluting substances may decrease or increase the ionisation of the analyte. Matrix effect depends mainly on the sample type, method of preparation, the quality of the chromatographic separation, the mobile phase additives, as well the method of sample ionization. Degree of enhancement or suppression of the ionization is also dependent on physicochemical properties of the analyte [1, 2]. There are two types of matrix effects: absolute and relative. The absolute matrix effect is defined as the difference between the signal response of the mass spectrometer to the analyte in the calibration solution and the same amount of analyte in a sample extract with a complex matrix, e.g. in a biological sample. There are two types of absolute matrix effects. One is the suppression of the ionization, which is most frequently observed, and the other is enhancement of the ionization. The relative matrix effect is a change of the absolute matrix effects between each batch of samples containing the same matrix. Indeed it was observed that the matrix effect of the same analyte in the same
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mobile phase may vary from sample to sample. The level of endogenous substances is different in samples from different individuals [3, 4]. For this reason, the analysis of biological samples is very complicated, because each sample is individual and the level of induced matrix effects may vary. Due to the fact that matrix compounds may adversely affect the important parameters and measurement performance of the methods they should be reduced or eliminated by optimizing the chromatographic conditions, purification of the sample, or even change the type of ionization. Components of the sample, which induce the matrix effect, can be removed during the sample preparation (preconcentration, purification) stage. Methods of preconcentration include protein precipitation (PPT), liquid–liquid extraction (LLE) or solid phase extraction (SPE). LLE and SPE are more laborious than PPT, but generally result in a better removal of the matrix components. Dilution of the sample is also a way to effectively minimize matrix effects. However, dilution also decreases the concentration of analyte in the sample [5]. It is not always possible to completely eliminate the matrix effect. Above mentioned sample preparations remove the major part of the endogenous material, but a small amount often remains in the sample and still can induce matrix effect. For this reason, it must be monitored, and it is important to determine its magnitude. The estimation of the strength of matrix effect is a critical technical aspect of method development. Currently the evaluation of matrix effect is a mandatory part of LC–MS method validation, however there is no defined way to perform it [6, 7]. In order to examine the qualitative matrix effect, the postcolumn injection of analyte into the system, directly into the detector, is used. With this approach, qualitative identification of the regions on chromatogram where there is an increase or decrease of the signal is possible. In this technique the sample, subjected to an appropriate preparation (e.g. extraction), is injected into the chromatographic column, while a continuous stream of the analyte (tested substance) is injected through a tee between the column and the mass spectrometer source. In addition, the blank sample such as water, buffer or mixture of mobile phase (e.g. acetonitrile– water) is also injected into the column in order to establish a baseline for the analysis. Matrix compounds that elute from the column cause a variation in ionization response. Regions of the suppression or enhancement of the signal can be visualized by comparing the baseline chromatograms with each of the tested matrices. The permanent postcolumn infusion of the analyte provides information on matrix effects occurring during a whole chromatographic run but it JOURNAL OF ANALYTICAL CHEMISTRY
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only qualitatively illustrates the degree of signal change. The first application of the aforementioned manner of matrix effect estimation was published by Bonfiglio et al. and since then it was widely used by other authors [8–10]. In order to quantify the matrix effect, postcolumn injection is not required. Most often, two sets of samples are used. One of them contains the analyte added to the beforehand extracted matrix, and the second contains the analyte dissolved in a buffer, solvent or mobile phase. Both sets contain the same amount of the analyte, and they are analyzed in the same way. The degree of suppression or enhancement is calculated using the following Matuszewski formula [2, 3]: Matrix effect (%) = B/A × 100%, where A is peak area of the analyte in the external solution (mobile phase buffer, etc.), and B is peak area of the analyte added to the matrix after extraction. Obtaining 100% indicates absence of any matrix effect, whereas <100% means suppression and values >100% indicate enhancement of the ionization process. Most researchers use this post-extraction method for evaluation of matrix effect; however, they often did not evaluate the effect of matrices from different sources [7, 11]. It should also be noted that some authors subtract number 1 from the peak area ratios, and then a zero value indicates lack of matrix effect, while negative values indicate suppression and positive enhancement [12, 13]. Both of the aforementioned methods for determining the matrix effect are characterized by advantages and disadvantages. Therefore, the aim of this study was to describe a method for determination of the matrix effect which would combine the advantages of both of the above methods while eliminating their disadvantages. In order to illustrate the method and to show its usefulness, synthetic cannabinoids were chosen and isolated from the blood in different ways. It should be noted, however, that the aim of the study was to show a simple method of determination of the matrix effect, rather than a thorough examination of matrix effects for these compounds. For the study, synthetic cannabinoids: (1-pentylindol3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone (UR144), [1-(5-fluoropentyl)-1H-indol-3-yl](2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11) and N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (STS-135), were used. Their chemical structures are presented below. Synthetic cannabinoids are components of so called ‘legal highs’ and are becoming increasingly popular.
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C H3 O
CH3 CH3
CH3 O
CH3
O
CH3
N CH3 UR-144
CH3 CH3
N XLR-11
EXPERIMENTAL Chemicals UR-144, STS-135 and XLR-11 standards were purchased from LGC Standards (Dziekanow Lesny, Poland). HPLC-grade acetonitrile (MeCN), ethyl acetate, isopropanol, methanol and formic acid (98– 100%) were bought from Merck (Warsaw, Poland). Biological Material Drug-free whole (blank) blood samples were obtained from a regional blood donation centre. Blank blood screens for common drugs of abuse and new psychoactive substances (including UR-144, STS-135 and XLR-11) were negative. Standards Preparation Stock solutions of synthetic cannabinoids (1 mg/mL in methanol) were stored below –20°C. Working solutions were prepared before analysis at concentration of 5 μg/mL in water. Matrix Preparation The blood samples for testing were prepared in different ways. The first was liquid–liquid extraction at pH 9, the second was solid phase extraction, and the last was precipitation with acetonitrile. The individual protocols are shown below. The prepared samples were stored at –20°C. Liquid–liquid extraction. To the 0.4 mL of blood samples placed in Eppendorf vials, 0.4 mL of carbonate buffer and 1 mL of ethyl acetate were added. The samples were mixed for 10 min and centrifuged at 13000 rpm (15.682 g) for 5 min. The organic layers (0.8 mL) were then transferred to 2-mL glass vials and evaporated to dryness. The dry residues were dissolved in 200 μL of the mixture of 0.1% formic acid in water and 0.1% formic acid in MeCN (1 : 1, v/v) and transferred to microvolume inserts for autosampler vials (allowing low volume sampling). Solid phase extraction. To the 0.4 mL of blood samples placed in Eppendorf vials, 1 mL of deionized
F
NH
F
N STS-135
water was added, the samples were mixed for 5 s and centrifuged at 13000 rpm for 5 min. The Oasis HLB SPE column was preconditioned with methanol (2 mL) and deionized water (2 mL). The sample was transferred to the column and slowly passed through it (under small vacuum) followed by rinsing with 2 mL of deionized water. The column bed was dried under a full vacuum for 5 min. The elution was performed with 0.6 mL of methanol and 0.6 mL of a mixture of methanol–isopropanol (3 : 1, v/v) under gravity. The eluate was evaporated to dryness, the dry residues were dissolved in 200 μL of the mixture of 0.1% formic acid in water and 0.1% formic acid in MeCN (1 : 1, v/v) and transferred to inserts for autosampler vials. Precipitation. To the 0.4 mL of blood sample placed in Eppendorf vial, 0.8 mL of MeCN was added in 50 μL portions, and after each addition the sample was vortex mixed for 10 s. The samples were mixed for 10 min and centrifuged at 13000 rpm for 5 min. The MeCN phase (0.8 mL) was then transferred to a 2-mL glass vial and evaporated to dryness. The dry residues were dissolved in 200 μL of mixture 0.1% formic acid in water and 0.1% formic acid in MeCN (1 : 1, v/v) and transferred to inserts for autosampler vials. Chromatographic and Spectrometric Conditions Analyses were performed on an Agilent-Hewlett Packard LC-MSD 1100 Series liquid chromatograph coupled to single quadrupole mass selective detector operating in electrospray positive ionization (+ESI) mode (Hewlett Packard; Perlan Technologies, Warsaw, Poland). Separation was achieved on a LiChroCART (125 × 3 mm) Purospher RP-18e (5 μm) column (Merck, Warsaw, Poland) maintained at 25°C. The mobile phase consisting of a mixture of 0.1% formic acid in MeCN (v/v) and 0.1% formic acid in water (v/v) was eluted under the following gradient conditions (shown in relation to MeCN content): 0 min— 0%, 6 min—100%, 11 min—0%, 19 min—0%. The mobile phase was delivered at a flow rate of 0.5 mL/min, and the total analytical run time was 19 min. The injection volume was 10 μL. Selected ion monitoring (SIM) was applied. The protonated molecule and fragment ions for each compound were mon-
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itored. The following ions were monitored (m/z): 125.2, 144.1, 214.1, 312.2, 313.2 for UR-144; 125.2, 144.1, 232.0, 330.2 for XLR-11 and 135.1, 144.1, 232.1, 327.2, 383.3 for STS-135. The mass detector parameters were as follows: capillary voltage 3500 V, fragmentor voltage 180 V, drying gas flow (nitrogen) 13 L/min, gas temperature 320°C, nebulizer pressure 30 psi. The apparatus maintenance and analysis of results were conducted using HP LC/MSD ChemStation. The post-column infusion was performed with the use of LEGATO 100 single syringe pump (KD Scientific; Bioeko, Krakow, Poland). The applied dosing rate was 10 μL/min, while the concentration was 5 μg/mL for all synthetic cannabinoids. RESULTS AND DISCUSSION LC–MS is extensively used for identification and quantification of different compounds, especially drugs and metabolites in biological fluids, however it is susceptible to matrix effects. ESI and atmospheric pressure chemical ionization (APCI) are used as the interfaces between LC and MS and these are places where matrix effect occurs. APCI causes less matrix effect because there is no competition between analytes to enter the gas phase [7, 10, 14]. Due to this fact that APCI is less susceptible to matrix effect than ESI, as well as that ESI is most commonly used, we decided to apply this type of ionisation in the whole study. The two main techniques used to determine the degree of matrix effect on an LC–MS method have advantages and disadvantages. The post-column infusion allows the identification of the regions over the entire chromatogram where there is an increase or decrease of the signal. This type of matrix effect evaluation and relative abundance comparison was presented by some authors [8, 10–12, 14]. This approach however is limited because direct comparison of chromatograms does not allow for precise quantification of the effect at a specific site of the chromatogram. In fact, it is a qualitative method. In turn, the postextraction addition technique, used by most researchers during method development, allows the accurate calculation of the effect but only at the point of elution of the analyte of interest. Using this method, we do not know the matrix effect in other areas of the chromatogram, in particular near the point of elution, which can be especially important when we take into account that matrix effect may also cause retention time drift and chromatographic peak tailing. So we can say that both techniques provide complementary information. In this context, we propose to use a method that connects the advantages of both of the above, while eliminating their disadvantages. The direct comparison of post-column infusion chromatograms has one more major drawback. The most important for the conducted experiments was reproducibility of the results, because matrix effects JOURNAL OF ANALYTICAL CHEMISTRY
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often tend to result in subtle differences that can not be noticed, when low reproducible results were obtained. Elements of the mass spectrometer are successively contaminated during the use as well as some parts are subject to wear. During the analyses the impurities accumulating in the ion source, as well as in capillary or on skimmers, may lead to lowering of the analytical signal. Similar effects are caused by a slow wear of the multiplier horn. Therefore, comparing the matrix effect profiles obtained over time can lead to erroneous results. The decrease in signal may be regarded as a matrix effect while it can be caused by the contamination of the apparatus. Therefore, application of this technique seems to have limited applicability, in particular for long-term experiments. The problems with the stability of the system and thus the reproducibility of the results were observed during our preliminary research. Despite the greatest care and obtaining stable signals and comparable chromatograms within one to several days, it was not possible to accurately compare all the results (chromatograms) obtained for several months of research. For this reason, we decided to use normalized matrix effect profiles and compare not the same chromatograms but matrix effects in the entire range of the run. Practically data from each apparatus can be exported as a txt, csv or xls files. Approximately 2700 data points were obtained during 19 min run of the applied method (one intensity reading point for less than 0.01 min). These data were inserted to and processed in Excel spreadsheets. The measurements for each injection were repeated several times (at least 5) and the intensity readings were averaged. The use of a spreadsheet allowed easy calculation of the matrix effect in accordance with the Matuszewski formula for each measurement point. The results obtained for extracted and blank samples only in one day, under identical conditions were used to create a matrix profile. Each experiment was performed in 5–10 repetitions. Obtained in this way, normalized matrix effect profiles were very reproducible and allowed to compare them even for experiments carried out over time. The matrix effect data for a large number of points allowed precise plotting normalized profiles for the entire chromatograms. In this way it was possible to combine the two most common methods of determining of matrix effect and thus to obtain quantitative results throughout the whole run time range. The presented procedure seems simple, but is so far not widely presented in the scientific papers. The study confirming the practical applicability of the developed method was focused on the synthetic cannabinoids. This group was chosen because the increasing number of new psychoactive substances, including synthetic cannabinoids, is a significant problem for toxicologists. Users of synthetic cannabinoids often associate this group of compounds with marijuana, while their use carries a number of risks. In recent years, more and more reports of poisonings
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with synthetic cannabinoids appear in the professional literature [15–18]. Expected concentrations of these substances in the blood are low, so the impact of possibly occurring matrix effects can be important for the results of their determinations. The analyses of matrix effect were conducted for different sample preparation methods (including PPT with acetonitrile, SPE and LLE from a pH 9). Various techniques of sample preparation are available but these are widely used. Each injection was repeated at least five times to obtain reliable and comparable chromatograms. Before the relevant experiments on the matrix effect for each analyte, a number of studies with different fragmentor voltages (in a range of 90–190 V) were performed. It allowed to select the optimum voltage at which there was a suitable fragmentation of analytes, and thus to monitor various fragment ions, not only the protonated molecule. These experiments showed that the optimal fragmentor voltage value was 180 V. The voltages below this value lead to soft fragmentation of the analytes. The choice of higher fragmentation voltage, and thus the monitoring of the spectrum rich in fragment ions, allowed later to investigate the dependence of the matrix effect on the monitored ion. The matrix effect was tested using the technique involving the injection of a properly prepared blood sample (free of the tested substance) and then the blank sample (mixture of water and MeCN, 1 : 1, v/v) onto a chromatographic column with continuous post-column infusion of the standard (UR-144, XLR-11 or STS-135). Next, the matrix effect was calculated in the spreadsheets and the normalized chromatographic profiles of the different samples were drawn and compared. An important aspect in the analysis of the matrix effect is proper concentration of the analyte in the sample. Too high concentration may result in masking of this effect. All regions of suppression or enhancement of the signal should be compared with the region close to the retention time of the analyte. The rate of post-column standard dosage and concentration were selected on the basis of the available literature and preliminary experiments. The concentration of the analyte should be in the analytical range being investigated. The flow of 10 μL/min and the concentration of standard 5 μg/mL made it up that in a few dozen seconds (the time corresponding to the width of a typical chromatographic peak) the detector reached about several to tens of ng of the substance. It can therefore be concluded that the studies were carried out in the range of the real concentrations of synthetic cannabinoids that may be found in the blood. In Fig. 1, an example comparison of the traditional method of presenting postcolumn injection chromatographic profiles according to Bonfiglio et al. and the method discussed by the authors is presented (both for the same data sets). It is clear that presenting
matrix effect in the method described in this work is much more useful. The upper figure allows only a subjective estimation of the matrix effect while the lower accurately represents its value. Conducted experiments allowed to assess the efficiency with which three common sample preparation techniques removed matrix components from blood. Of course, the results relate only to the analyzed synthetic cannabinoids and only to matrix effect. The recovery experiments were not conducted, and you may find that this factor may be crucial, and more important than matrix effect, in choosing the method of sample preparation. When analyzing the obtained normalized matrix effect profiles for synthetic cannabinoids, a few conclusions may be drawn. Large differences in matrix effect were observed between sample preparation techniques. Significantly smaller reduction of the signal was observed for LLE samples in relation to the samples PPT with acetonitrile (Fig. 2). This was also observed for the SPE; however, this type of sample preparation was characterized by an intermediate degree of suppression. In general, acetonitrile PPT had the greatest matrix effect. These findings were consistent with those obtained by other authors [7, 8]. This confirms the fact that during precipitation with acetonitrile not only analytes were isolated but also significant amounts of the matrix components. In some areas of chromatograms a decrease in the signal below 10% was observed for PPT (for STS-135 at RT ~1.8 min). However, it is common to see suppression of the signal at the time that corresponds to the void volume of the column [19]. Around the column dead volume poorly retained polar analytes are eluted. The application of LLE from an alkaline medium (pH 9) resulted in the slightest effect for UR-144 on the analytical signal. In the considerably larger part of the profile the matrix effect was maintained around 80– 100%. For XLR-11 and STS-135 the suppression after LLE was up to 60%. Nevertheless, even in this case it cannot be concluded that this method of sample preparation completely eliminates matrix effects. Other authors also observed large differences in matrix effect between sample preparation techniques. The acetonitrile PPT had the greatest matrix effect with major suppression areas in the beginning and at the end of chromatographic separation [7, 12]. It may seem surprising that SPE was characterized by greater matrix effect than LLE while SPE is often proposed as a solution for matrix effect [7]. This can be explained by the use of the all purpose columns in the experiment. Oasis HLB are columns for acidic, basic and neutral compounds and such a wide spectrum can definitely cover a lot of matrix components. When analyzing the data individually, a significant differences for each compound (UR-144, STS-135 and XLR-11) can be seen. For the XLR-11 differences between the profiles for different methods of preparation were least of all. The course of the matrix effect profile for the SPE and LLE were almost identical.
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(a)
Mobile phase
Analyte peak (XLR-11)
Abundance
LLE (pH 9)
SPE
PPT
Retention time, min (b) 200 180 160
LLE (pH 9)
Matrix effect, %
140 120 100 80 60 40 PPT
20 SPE 0
2
4
6
8 10 12 Retention time, min
14
16
18
Fig. 1. Comparison of postcolumn infusion of XLR-11 for different methods of sample preparation: chromatograms profiles according to Bonfiglio et al. [8] (a) and normalized matrix effect profiles used by authors (b).
However, in the case of STS-135 these profiles had significantly different course. The differences in matrix effect were even 80% in most of continuity of the profiles. Monitoring not only the protonated molecule but also fragment ions showed that the matrix effect was also dependent on the ion which was monitored. In the case of UR-144 the differences between profiles were not too large and for the sample prepared by LLE JOURNAL OF ANALYTICAL CHEMISTRY
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they were practically unnoticeable. In the other two methods of sample preparation the differences were greater, both for different ions and for different methods of sample preparation. It should be pointed out that without the use of, as described in this work, the normalized chromatographic matrix effect profiles it would not be possible to compare different ions. This is due to the considerable differences in their intensities. Comparison of normalized matrix effect profiles
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200 180 160 140 120 100 80 60 40 20
Matrix effect, %
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Matrix effect, %
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UR-144 (SIM, pseudomolecular ion m/z = 312)
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SPE PPT
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4
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SPE
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18
STS-135 (SIM, pseudomolecular ion m/z = 383)
LLE (pH 9)
SPE
PPT
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4
6 8 10 12 14 Retention time, min
16
18
Fig. 2. Comparison of normalized matrix effect profiles of synthetic cannabinoids for samples prepared in different ways.
of STS-135 for samples prepared in different ways for different ions are presented in Fig. 3. Lack of recovery determination makes it impossible to determine whether shown results were caused only by the matrix effect affecting the ionization effi-
ciency of the analytes or by these effects along with the recovery of analytes. Therefore, the obtained results should be treated very carefully, and they certainly may pose a topic for further research. It should be pointed that the use of internal standards, in particular
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STS-135 − LLE (pH 9)
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5 0
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Fig. 3. Comparison of normalized matrix effect profiles of STS-135 for samples prepared in different ways for different ions with m/z 383 (1), 307 (2), 232 (3), 144 (4) and 135 (5).
deuterated derivatives, would greatly reduce the adverse effect of the matrix. To summarize the above it is clear that the use of normalized matrix effect profiles considerably facilitates the tracing of analytical signal intensity changes. Matrix effects for synthetic cannabinoids have not been widely discussed here since the main purpose of this paper is to show the utility of the discussed matrix effect evaluation technique. The use of normalized JOURNAL OF ANALYTICAL CHEMISTRY
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profiles in determining the matrix effect on the example of synthetic cannabinoids perfectly demonstrated the practical uses of such a method. Simple transfer of data to common spreadsheets allowed for easy and accurate calculation of matrix effect throughout the entire chromatographic run. Understanding the matrix effect throughout the chromatogram can greatly facilitate the process of optimization of the method. It also should be noted,
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however, that more important than the matrix effect over the entire chromatogram is the matrix effect observed at or near the retention time of the tested substance. Retention times for UR-144, XLR-11 and STS-135 in the described conditions were 11.12, 12.48 and 12.46 min, respectively, and these retention times should be the most important in the context of the matrix effect examination. Even large changes in signal intensity are less important during the analysis if it does not refer to the retention time of eluted compound. Efforts should be made to create such a method that the substance is analyzed in a retention time where the matrix effect is minimal. However, it is very difficult. The variation of gradient profile will change the ionization conditions due to change of electrospray composition. It concerns both matrix compounds and components of the eluent. Consequently, the matrix effects will also change. However, the proposed technique for determining the matrix effect also allows getting from a spreadsheet specific matrix effect values for a specific retention time. We present an alternative way of estimation of matrix effect compiling the two most popular methods—qualitative and quantitative. This method for determination of matrix effect combines the advantages of aforementioned traditional methods while eliminating their disadvantages. The discussed approach was applied for matrix effect evaluation for synthetic cannabinoids. Results indicated that matrix effect for these compounds was highly dependent on sample preparation. Although similar structure significant differences were observed for different compounds. The experiments described in this paper and their results showed that such method of matrix effect estimation can have practical applications in the development of analytical methods. The use of this approach can help to modify chromatographic conditions to obtain the lowest matrix effect. The comparison of the normalized matrix effect profiles can be done even for data obtained within distant time.
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