{"id":43559,"date":"2021-01-12T13:30:00","date_gmt":"2021-01-12T13:30:00","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/fast-direct-analysis-of-underivatized-amino-acids-in-infant-formula\/"},"modified":"2026-03-17T21:28:24","modified_gmt":"2026-03-17T21:28:24","slug":"fast-direct-analysis-of-underivatized-amino-acids-in-infant-formula","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/zh-hans\/articles\/fffa3436\/fast-direct-analysis-of-underivatized-amino-acids-in-infant-formula","title":{"rendered":"Fast, Direct Analysis of Underivatized Amino Acids in Infant Formula"},"content":{"rendered":"\n<ul class=\"wp-block-list\">\n<li>Simple, one-step sample preparation for direct analysis of underivatized amino acids.<\/li>\n\n\n\n<li>Simultaneous analysis of nonpolar, polar, positively charged, and negatively charged amino acids in a short 10-minute run.<\/li>\n\n\n\n<li>Well-designed gradient separates contaminants from target analytes.<\/li>\n<\/ul>\n\n\n\n<p>To ensure that nutritional requirements are met, reliable analytical methods are needed for the accurate determination of free amino acids in infant formulas. Precolumn derivatization followed by reversed-phase LC analysis is a typical approach, with derivatization being necessary due to the lack of chromatographic retention and poor sensitivity for free amino acids. However, these methods are often time-consuming and labor intensive. The use of perfluorinated acids as ion-pairing reagents to improve the retention of underivatized amino acids on a C18 column is another common technique, but this practice can negatively impact the chromatographic system and mass spectrometer.<\/p>\n\n\n\n<p>In the simpler approach shown here, underivatized amino acids can be measured directly\u2014following a single-step sample preparation procedure\u2014using a Raptor Polar X column paired with an MS\/MS detector. Raptor Polar X columns feature a hybrid phase chemistry (HILIC and ion exchange) that provides the balanced retention needed for simultaneous analysis of a wide range of analyte chemistries. As demonstrated, underivatized amino acids with nonpolar, polar, positively charged, and negatively charged side chains were adequately retained and then quickly eluted using a 10-minute gradient. This gradient also separated amino acid system contaminants from target analytes, adding ruggedness to the method. By following this method, time- and labor-intensive sample preparation procedures, which can include expensive derivatization kits, are replaced by a simple protein precipitation and direct analysis of the resulting extract. Direct analysis of underivatized amino acids on a Raptor Polar X column provides excellent results in a fast, easy workflow, making this a beneficial alternative to traditional methods.<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><\/div><div class='chromatogram-article-inner-full'><div class=\"chromatogram-article-inner\">\n<div class=\"wp-block-custom-chromatogram-article\"><div class=\"wp-block-custom-chromatogram-article\"><div class=\"chromatogram-image regular-image\"><img decoding=\"async\" src=\"https:\/\/ez.restek.com\/images\/cgram\/lc_ff0579.png\" alt=\"Underivatized Amino Acids Analysis in Baby Formula on Raptor Polar X\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_FF0579<\/p><div class=\"chromatogram-peaks\"><h4>Peaks<\/h4><table class=\"peaks col-lg-6 col-12 peak-50\">\n<thead><tr><th><\/th><th style=\"text-align: left;width: 75px\">Peaks<\/th><th style=\"text-align: center;width: 75px\">t<sub>R<\/sub> (min)<\/th><th style=\"text-align: center;width: 75px\">Precursor Ion<\/th><th style=\"text-align: center;width: 75px\">Product Ion<\/th><\/tr><\/thead>\n<tbody><tr><td class=\"num\">1.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Tryptophan\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/54-12-6\/Tryptophan\" target=\"_blank\" rel=\"noopener\">Tryptophan<\/a><\/td><td class=\"oth\">1.17<\/td><td class=\"oth\">205.07<\/td><td class=\"oth\">146.08<\/td><\/tr>\n<tr><td class=\"num\">2.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Phenylalanine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/63-91-2\/Phenylalanine\" target=\"_blank\" rel=\"noopener\">Phenylalanine<\/a><\/td><td class=\"oth\">1.26<\/td><td class=\"oth\">166.13<\/td><td class=\"oth\">120.10<\/td><\/tr>\n<tr><td class=\"num\">3.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Leucine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/61-90-5\/Leucine\" target=\"_blank\" rel=\"noopener\">Leucine<\/a><\/td><td class=\"oth\">1.41<\/td><td class=\"oth\">132.13<\/td><td class=\"oth\">86.10<\/td><\/tr>\n<tr><td class=\"num\">4.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Isoleucine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/73-32-5\/Isoleucine\" target=\"_blank\" rel=\"noopener\">Isoleucine<\/a><\/td><td class=\"oth\">1.55<\/td><td class=\"oth\">132.13<\/td><td class=\"oth\">86.10<\/td><\/tr>\n<tr><td class=\"num\">5.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Methionine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/59-51-8\/Methionine\" target=\"_blank\" rel=\"noopener\">Methionine<\/a><\/td><td class=\"oth\">1.62<\/td><td class=\"oth\">150.07<\/td><td class=\"oth\">104.10<\/td><\/tr>\n<tr><td class=\"num\">6.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Tyrosine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/556-03-6\/Tyrosine\" target=\"_blank\" rel=\"noopener\">Tyrosine<\/a><\/td><td class=\"oth\">1.69<\/td><td class=\"oth\">182.10<\/td><td class=\"oth\">136.08<\/td><\/tr>\n<tr><td class=\"num\">7.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Taurine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/107-35-7\/Taurine\" target=\"_blank\" rel=\"noopener\">Taurine<\/a><\/td><td class=\"oth\">1.91<\/td><td class=\"oth\">126.07<\/td><td class=\"oth\">108.07<\/td><\/tr>\n<tr><td class=\"num\">8.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Valine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/516-06-3\/Valine\" target=\"_blank\" rel=\"noopener\">Valine<\/a><\/td><td class=\"oth\">1.98<\/td><td class=\"oth\">118.13<\/td><td class=\"oth\">72.11<\/td><\/tr>\n<tr><td class=\"num\">9.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Proline\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/609-36-9\/Proline\" target=\"_blank\" rel=\"noopener\">Proline<\/a><\/td><td class=\"oth\">2.29<\/td><td class=\"oth\">116.13<\/td><td class=\"oth\">70.09<\/td><\/tr>\n<tr><td class=\"num\">10.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Alanine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/302-72-7\/Alanine\" target=\"_blank\" rel=\"noopener\">Alanine<\/a><\/td><td class=\"oth\">3.00<\/td><td class=\"oth\">90.03<\/td><td class=\"oth\">44.10<\/td><\/tr>\n<\/tbody><\/table>\n<table class=\"peaks col-lg-6 col-12 peak-50\"><thead><tr><th><\/th><th style=\"text-align: left;width: 75px\">Peaks<\/th><th style=\"text-align: center;width: 75px\">t<sub>R<\/sub> (min)<\/th><th style=\"text-align: center;width: 75px\">Precursor Ion<\/th><th style=\"text-align: center;width: 75px\">Product Ion<\/th><\/tr><\/thead><tbody>\n<tr><td class=\"num\">11.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Threonine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/80-68-2\/Threonine\" target=\"_blank\" rel=\"noopener\">Threonine<\/a><\/td><td class=\"oth\">3.12<\/td><td class=\"oth\">120.13<\/td><td class=\"oth\">74.08<\/td><\/tr>\n<tr><td class=\"num\">12.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Glycine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/56-40-6\/Glycine\" target=\"_blank\" rel=\"noopener\">Glycine<\/a><\/td><td class=\"oth\">3.62<\/td><td class=\"oth\">76.10<\/td><td class=\"oth\">30.11<\/td><\/tr>\n<tr><td class=\"num\">13.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Glutamine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/56-85-9\/Glutamine\" target=\"_blank\" rel=\"noopener\">Glutamine<\/a><\/td><td class=\"oth\">3.87<\/td><td class=\"oth\">147.13<\/td><td class=\"oth\">84.07<\/td><\/tr>\n<tr><td class=\"num\">14.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Serine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/302-84-1\/Serine\" target=\"_blank\" rel=\"noopener\">Serine<\/a><\/td><td class=\"oth\">3.93<\/td><td class=\"oth\">106.07<\/td><td class=\"oth\">60.09<\/td><\/tr>\n<tr><td class=\"num\">15.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Asparagine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/70-47-3\/Asparagine\" target=\"_blank\" rel=\"noopener\">Asparagine<\/a><\/td><td class=\"oth\">4.08<\/td><td class=\"oth\">133.13<\/td><td class=\"oth\">74.07<\/td><\/tr>\n<tr><td class=\"num\">16.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Arginine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/74-79-3\/Arginine\" target=\"_blank\" rel=\"noopener\">Arginine<\/a><\/td><td class=\"oth\">4.47<\/td><td class=\"oth\">175.17<\/td><td class=\"oth\">70.09<\/td><\/tr>\n<tr><td class=\"num\">17.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Histidine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/71-00-1\/Histidine\" target=\"_blank\" rel=\"noopener\">Histidine<\/a><\/td><td class=\"oth\">4.66<\/td><td class=\"oth\">156.07<\/td><td class=\"oth\">110.16<\/td><\/tr>\n<tr><td class=\"num\">18.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Lysine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/56-87-1\/Lysine\" target=\"_blank\" rel=\"noopener\">Lysine<\/a><\/td><td class=\"oth\">4.97<\/td><td class=\"oth\">147.13<\/td><td class=\"oth\">84.13<\/td><\/tr>\n<tr><td class=\"num\">19.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Glutamic acid\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/617-65-2\/Glutamic acid\" target=\"_blank\" rel=\"noopener\">Glutamic acid<\/a><\/td><td class=\"oth\">5.89<\/td><td class=\"oth\">148.10<\/td><td class=\"oth\">84.10<\/td><\/tr>\n<tr><td class=\"num\">20.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Cystine\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/56-89-3\/Cystine\" target=\"_blank\" rel=\"noopener\">Cystine<\/a><\/td><td class=\"oth\">6.10<\/td><td class=\"oth\">241.13<\/td><td class=\"oth\">152.00<\/td><\/tr>\n<tr><td class=\"num\">21.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Aspartic acid\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/617-45-8\/Aspartic acid\" target=\"_blank\" rel=\"noopener\">Aspartic acid<\/a><\/td><td class=\"oth\">7.12<\/td><td class=\"oth\">134.07<\/td><td class=\"oth\">74.06<\/td><\/tr>\n<\/tbody><\/table><\/div><div class=\"chromatogram-conditions\"><h4>Conditions<\/h4><div class=\"conditions-container container-fluid\"><div class=\"row\"><table class=\"conditions col-lg-6 col-12\"><tr><th class=\"conditions_header\" scope=\"row\">Column<\/th><td>Raptor Polar X  (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9311A12?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_FF0579\" rel=\"noopener\">cat.# 9311A12<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Dimensions:<\/th><td>100 mm x 2.1 mm ID<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Particle Size:<\/th><td>2.7 \u00b5m<\/td><\/tr><tr><td><\/td><tr><th class=\"sub conditions_header\" scope=\"row\">Temp.:<\/th><td>30 \u00b0C<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Standard\/Sample<\/th><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Diluent:<\/th><td>20:80 Water:acetonitrile, 0.01 N HCl<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Conc.:<\/th><td>  Endogenous amino acids<\/td><\/tr><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Inj. Vol.:<\/th><td>5 \u00b5L <\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Mobile Phase<\/th><td><\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">A:<\/th><td>Water, 0.5% formic acid <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">B:<\/th><td>Mobile Phase B: 9:1 Acetonitrile:20 mM ammonium formate in water (pH 3.0) (The ammonium formate concentration is 20 mM relative to the total volume of mobile phase B. See preparation notes for instructions on diluting a 200 mM aqueous starting solution.) <\/td><\/tr><tr><td><\/td><td><table class=\"cgram_ramp\"><thead><tr><th>Time (min)<\/th><th>Flow (mL\/min)<\/th><th>%A<\/th><th>%B<\/th><\/tr><\/thead><tbody><tr><td>0.00<\/td><td>0.5<\/td><td>12<\/td><td>88<\/td><\/tr><tr><td>3.50<\/td><td>0.5<\/td><td>12<\/td><td>88<\/td><\/tr><tr><td>8.00<\/td><td>0.5<\/td><td>70<\/td><td>30<\/td><\/tr><tr><td>8.01<\/td><td>0.5<\/td><td>12<\/td><td>88<\/td><\/tr><tr><td>10.0<\/td><td>0.5<\/td><td>12<\/td><td>88<\/td><\/tr><\/tbody><\/table><\/td><\/tr><\/table><table class=\"conditions col-lg-6 col-12\"><tr><th class=\"conditions_header\" scope=\"row\">Detector<\/th><td>MS\/MS<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Ion Mode:<\/th><td>ESI+ <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Mode:<\/th><td>MRM <\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Instrument<\/th><td>UHPLC<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Sample Preparation<\/th><td>A 200 \u00b5L aliquot of protein hydrolysate formula (Similac ALIMENTUM) was mixed with 800 \u00b5L of acetonitrile and 10 \u00b5L of 1 N HCl. After centrifugation at 4000 rpm for 5 minutes, the supernatant was diluted 20-fold with 20:80 water:acetonitrile (0.01 N HCl) and injected for analysis.<\/td><\/tr><tr class=\"cgram_header_row\"><th class=\"conditions_header\" scope=\"row\">Notes<\/th><td><i>Mobile Phase B Preparation:<\/i> To make 500 mL of mobile phase B, measure ~45 mL of water into a small beaker and add 1 mL of 10 M ammonium formate solution. Adjust pH to 3.0 by adding formic acid and then bring the volume to 50 mL with water. Combine this 50 mL ammonium formate solution (pH 3.0) with 450 mL of acetonitrile to complete the preparation.   <\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_ff0579.pdf\" target=\"_blank\" rel=\"noopener noreferrer\"><svg xmlns=\"http:\/\/www.w3.org\/2000\/svg\" width=\"18\" height=\"18\" viewBox=\"0 0 18 18\"><g data-name=\"Group 2996\"><path data-name=\"Rectangle 1246\" d=\"M0 0h18v18H0z\" style=\"fill: none;\"><\/path><\/g><g data-name=\"Group 2997\"><path data-name=\"Path 729\" d=\"M13.412 11.4v2.017H5.345V11.4H4v2.017a1.349 1.349 0 0 0 1.345 1.345h8.068a1.349 1.349 0 0 0 1.345-1.345V11.4zm-.672-2.694-.948-.948-1.741 1.735V4H8.706v5.493L6.965 7.758l-.948.948 3.361 3.361z\" transform=\"translate(-.437 -.414)\" style=\"fill: rgb(13, 123, 196);\"><\/path><\/g><\/svg>Download PDF<\/a><\/div><\/div><\/div>\n<\/div><\/div><\/div>\n\n\n<div style=\"height:100px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n","protected":false},"excerpt":{"rendered":"<p>This direct LC-MS\/MS analysis of underivatized amino acids in infant formula is a simple workflow that provides good retention and fast elution of a wide range of amino acids.<\/p>\n","protected":false},"author":11,"featured_media":90018,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_kad_blocks_custom_css":"","_kad_blocks_head_custom_js":"","_kad_blocks_body_custom_js":"","_kad_blocks_footer_custom_js":"","_kadence_starter_templates_imported_post":false,"_kad_post_transparent":"","_kad_post_title":"","_kad_post_layout":"","_kad_post_sidebar_id":"","_kad_post_content_style":"","_kad_post_vertical_padding":"","_kad_post_feature":"","_kad_post_feature_position":"","_kad_post_header":false,"_kad_post_footer":false,"footnotes":""},"categories":[7],"tags":[],"industries-application":[2167,2168],"post-badge":[],"resource-type":[2623],"product-library":[2391,2373],"resource-technique":[2299,2302],"ppma_author":[414],"class_list":["post-43559","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-articles","industries-application-food-beverage","industries-application-food-quality","resource-type-featured-application","product-library-lc-columns","product-library-liquid-chromatography-products","resource-technique-liquid-chromatography","resource-technique-ms-ms"],"acf":[],"taxonomy_info":{"category":[{"value":7,"label":"Articles"}],"industries-application":[{"value":2167,"label":"Food &amp; 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