{"id":25,"date":"2024-04-04T00:39:46","date_gmt":"2024-04-03T21:39:46","guid":{"rendered":"https:\/\/sisu.ut.ee\/lcms_method_validation\/23-selectivity-examples\/"},"modified":"2024-04-15T11:38:27","modified_gmt":"2024-04-15T08:38:27","slug":"23-selectivity-examples","status":"publish","type":"page","link":"https:\/\/sisu.ut.ee\/lcms_method_validation\/23-selectivity-examples\/","title":{"rendered":"2.3. Selectivity examples"},"content":{"rendered":"

Example 1<\/strong><\/h2>\n\n\n\n

Not all validation guidelines explicitly specify a required limit for chromatographic peak resolution (R<\/em>S<\/sub>). In LC-MS an incomplete chromatographic separation of analyte peak from neighbouring peaks may or may not cause problems. Often m<\/em>\/z<\/em> of analyte differs from that\u00a0of neighbouring peaks or an unique MRM transition is used. This way the presence of any potentially overlapping peak is simply neglected. However, even though the analytical signals of these interfering compounds are not registered, they can cause matrix effects. Therefore, it is customary to seek for a peak resolution of at least R<\/em>S<\/sub> \u2265 1.5.<\/p>\n\n\n\n

The chromatogram in Figure 1 was recorded while developing a method for analysis of\u00a0compounds A, B, C and D. One MRM transition\u00a0is presented for each compound and is shown in different colour. If one hadn\u2019t recorded the transition shown in green, this peak would have been missing\u00a0from the chromatogram and one wouldn\u2019t have known that compound C (RT 5.33 min) \u2013 is present. But this compound could interfere with the detection of other compounds, regardless of whether or not it\u2019s signal is recorded. Therefore, one should make sure that the peak is adequately separated from other peaks.<\/p>\n\n\n\n

\"image001.png\"<\/figure>\n\n\n\n

<\/p>\n\n\n\n

Figure 1. Initial chromatographic separation of\u00a0compounds A, B, C and D.<\/h4>\n\n\n\n

Table 1 presents the chromatographic data for above separation. The peak resolution (R<\/em>S<\/sub>) from the preceding peak was calculated for the last 3 peaks according to equation 1. It appears that while the separation of the compounds B and C is at the limit (R<\/em>S<\/sub>= 1.5), then the separation of C and D is not satisfactory (R<\/em>S<\/sub> = 1.4).<\/p>\n\n\n\n

\"image003.png\"\u00a0 \u00a0 \u00a0 \u00a0 \u00a0 (Eq 1)<\/h6>\n\n\n\n

\u00a0<\/h4>\n\n\n\n

Table 1. Peak data of the chromatogram in Figure 1.<\/h4>\n\n\n\n
Analyte<\/strong><\/td>

t<\/em>R<\/sub><\/strong><\/p><\/td>

w<\/em>1\/2<\/sub><\/strong><\/p><\/td>

R<\/em>S<\/sub><\/strong><\/p><\/td><\/tr>

A<\/strong><\/td> 4.710<\/span><\/td>0.079<\/span><\/td><\/td><\/tr>
B<\/strong><\/td>5.102<\/span><\/td>0.094<\/span><\/td>2.7<\/span><\/td><\/tr>
C<\/strong><\/td>5.327<\/span><\/td>0.088<\/span><\/td>

<\/p>1.5<\/td><\/tr>

D<\/strong><\/td>5.538<\/span><\/td>0.093<\/span><\/td>1.4<\/span><\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n(1)<\/a>\n\n\n\n

The chromatographic separation was further optimized <\/p>\n\n\n\n

\u00a0and the chromatogram presented in Figure 2 was obtained. As a result, sufficient peak resolution (R<\/em>S<\/sub> > 1.5) has been\u00a0achieved for all of the analytes (Table 2).<\/p>\n\n\n\n

\"image003.png\"<\/figure>\n\n\n\n

<\/p>\n\n\n\n

Figure 2. Chromatographic separation of compounds A, B, C and D.<\/h4>\n\n\n\n

Table 2. Peak data of the chromatogram in Figure 2.<\/h4>\n\n\n\n
Analyte<\/strong><\/td> t<\/em>R<\/sub><\/strong><\/td> w<\/em>1\/2<\/sub><\/strong><\/td> R<\/em>S<\/sub><\/strong><\/td><\/tr>
A<\/strong><\/td> 4.752<\/span><\/td> 0.075<\/span><\/td>\u00a0<\/td><\/tr>
B<\/strong><\/td> 5.169<\/span><\/td> 0.074<\/span><\/td> 3.3<\/span><\/td><\/tr>
C<\/strong><\/td> 5.533<\/span><\/td> 0.077<\/span><\/td> 2.8<\/span><\/td><\/tr>
D<\/strong><\/td> 5.856<\/span><\/td> 0.088<\/span><\/td> 2.3<\/span><\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n

Example 2<\/strong><\/h2>\n\n\n\n

As seen in the previous example, while respecting the selectivity-enhancing power of MS, one must not forget the importance of the chromatographic separation. Chromatography becomes crucial if discrimination of isomers \u2013 having the same molecular mass \u2013 is desired.<\/p>\n\n\n\n

For example, two major metabolites of morphine are morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) (Figure 3).<\/p>\n\n\n\n

\"image004.png\"<\/figure>\n\n\n\n

<\/p>\n\n\n\n

Figure 3. Morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G).<\/h4>\n\n\n\n

M3G and M6G are structural isomers with identical molecular formulae and, consequently, identical molecular masses. In such situation, the\u00a0discrimination of isomers relies completely on chromatographic separation. A mass spectrometer, even if is a high resolution (HRMS) type, can\u2019t distinguish molecules of identical masses and fragmentation patterns (which is the case of these two compounds). However,\u00a0M3G and M6G can be conveniently separated chromatographically (Figure 4).<\/p>\n\n\n\n

\"image005.png\"<\/figure>\n\n\n\n

Figure 4.\u00a0Chromatographic separation of morphine metabolites Morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G).<\/h4>\n\n\n\n

***
(1) There are several options for selectivity optimization in reversed phase LC:<\/p>\n\n\n\n

    \n
  • Percent of organic solvent in the mobile phase<\/li>\n\n\n\n
  • Organic solvent itself (methanol, acetonitrile, THF or their mixture)<\/li>\n\n\n\n
  • pH of eluent (note the pH limits of your column; in case of LC-MS only volatile pH-buffer components can be used)<\/li>\n\n\n\n
  • Different column chemistry and physical properties<\/li>\n\n\n\n
  • Temperature (the effect is usually small, but may help)<\/li>\n<\/ul>\n","protected":false},"excerpt":{"rendered":"

    Example 1 Not all validation guidelines explicitly specify a required limit for chromatographic peak resolution (RS). In LC-MS an incomplete chromatographic separation of analyte peak from neighbouring peaks may or may not cause problems. Often m\/z of analyte differs from …<\/p>\n","protected":false},"author":60,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"inline_featured_image":false,"footnotes":""},"class_list":["post-25","page","type-page","status-publish","hentry"],"acf":[],"_links":{"self":[{"href":"https:\/\/sisu.ut.ee\/lcms_method_validation\/wp-json\/wp\/v2\/pages\/25","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/sisu.ut.ee\/lcms_method_validation\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/sisu.ut.ee\/lcms_method_validation\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/sisu.ut.ee\/lcms_method_validation\/wp-json\/wp\/v2\/users\/60"}],"replies":[{"embeddable":true,"href":"https:\/\/sisu.ut.ee\/lcms_method_validation\/wp-json\/wp\/v2\/comments?post=25"}],"version-history":[{"count":12,"href":"https:\/\/sisu.ut.ee\/lcms_method_validation\/wp-json\/wp\/v2\/pages\/25\/revisions"}],"predecessor-version":[{"id":1239,"href":"https:\/\/sisu.ut.ee\/lcms_method_validation\/wp-json\/wp\/v2\/pages\/25\/revisions\/1239"}],"wp:attachment":[{"href":"https:\/\/sisu.ut.ee\/lcms_method_validation\/wp-json\/wp\/v2\/media?parent=25"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}