{"id":43348,"date":"2020-10-15T14:30:00","date_gmt":"2020-10-15T14:30:00","guid":{"rendered":"https:\/\/discover.restek.com\/uncategorized\/analysis-of-fentanyl-and-its-analogues-in-human-urine-by-lc-msms\/"},"modified":"2026-01-28T16:28:04","modified_gmt":"2026-01-28T16:28:04","slug":"analysis-of-fentanyl-and-its-analogues-in-human-urine-by-lc-msms","status":"publish","type":"post","link":"https:\/\/discover.restek.com\/ja\/application-notes\/cfan2820\/analysis-of-fentanyl-and-its-analogues-in-human-urine-by-lc-msms","title":{"rendered":"Analysis of Fentanyl and Its Analogues in Human Urine by LC-MS\/MS"},"content":{"rendered":"\n<h2 class=\"wp-block-heading\">Abstract<\/h2>\n\n\n\n<p>Synthetic opioid drugs, such as fentanyl and sufentanil, have very high analgesic potency. Abuse of these prescription painkillers\u2014along with a rapidly growing list of illicit analogues\u2014is a significant public health problem. In this study, we developed a simple dilute-and-shoot method that provides a fast 3.5 minute analysis of fentanyl and related compounds (norfentanyl, acetyl fentanyl, alfentanil, butyryl fentanyl, carfentanil, remifentanil, and sufentanil) in human urine by LC-MS\/MS using a Raptor Biphenyl column.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Introduction<\/h2>\n\n\n\n<p>In recent years, the illicit use of synthetic opioids has skyrocketed, and communities worldwide are now dealing with an ongoing epidemic. Of the thousands of synthetic opioid overdose deaths per year, most are related to fentanyl and its analogues. With their very high analgesic properties, synthetic opioid drugs such as fentanyl, alfentanil, remifentanil, and sufentanil are potent painkillers that have valid medical applications; however, they are also extremely addictive and are targets for abuse. For example, carfentanil is a very powerful anesthetic used as a tranquilizer for large animals, primarily elephants. It is 10,000 times more potent than morphine, making it one of the most powerful synthetic opioids available. The increase in its illicit use, most commonly by mixing with heroin, has been linked to a significant number of overdose deaths since 2016. In addition to abuse of these prescription drugs, the current opioid crisis is fueled by a growing number of illicit analogues, such as acetyl fentanyl and butyryl fentanyl, which have been designed specifically to evade prosecution by drug enforcement agencies.<\/p>\n\n\n\n<p>As the number of opioid drugs and deaths increases, so does the need for a fast, accurate method for the simultaneous analysis of fentanyl and its analogues. Therefore, we developed this LC-MS\/MS method for measuring fentanyl, six analogues, and one metabolite (norfentanyl) in human urine (Figure 1). A simple dilute-and-shoot sample preparation procedure was coupled with a fast (3.5 minutes) chromatographic analysis using a Raptor Biphenyl column. This method provides accurate, precise identification and quantitation of fentanyl and related compounds, making it suitable for a variety of testing applications including clinical toxicology, forensic analysis, workplace drug testing, and pharmaceutical research.<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 1:<\/strong>\u00a0Chemical Structures \u00a0<\/div><div class='chromatogram-article-inner-full'><div class=\"chromatogram-article-inner\">\n<style>.kb-image43348_d14d79-6a .kb-image-has-overlay:after{opacity:0.3;}<\/style>\n<div class=\"wp-block-kadence-image kb-image43348_d14d79-6a\"><figure class=\"aligncenter size-large\"><img loading=\"lazy\" decoding=\"async\" width=\"1024\" height=\"788\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN2820-01-1024x788.jpg\" alt=\"\" class=\"kb-img wp-image-18928\" title=\"-\" srcset=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN2820-01-1024x788.jpg 1024w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN2820-01-300x231.jpg 300w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN2820-01-768x591.jpg 768w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN2820-01-1536x1182.jpg 1536w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN2820-01.jpg 1800w\" sizes=\"auto, (max-width: 1024px) 100vw, 1024px\" \/><\/figure><\/div>\n\n<\/div><\/div><\/div>\n\n\n<div style=\"height:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Experimental<\/h2>\n\n\n\n<h3 class=\"wp-block-heading\">Sample Preparation<\/h3>\n\n\n\n<p>The analytes were fortified into pooled human urine. An 80 \u00b5L urine aliquot was mixed with 320 \u00b5L of 70:30 water:methanol solution (five-fold dilution) and 10 \u00b5L of internal standard (40 ng\/mL in methanol) in a Thomson SINGLE StEP filter vial (Restek&nbsp;cat.# 25895). After filtering through the 0.2 \u00b5m PVDF membrane, 5 \u00b5L was injected into the LC-MS\/MS.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\">Calibration Standards and Quality Control Samples<\/h3>\n\n\n\n<p>The calibration standards were prepared in pooled human urine at 0.05, 0.10, 0.25, 0.50, 1.00, 2.50, 5.00, 10.0, 25.0, and 50.0 ng\/mL. Three levels of QC samples (0.75, 4.0, and 20 ng\/mL) were prepared in urine for testing accuracy and precision with established calibration standard curves. Recovery analyses were performed on three different days. All standards and QC samples were subjected to the sample preparation procedure described above.<\/p>\n\n\n\n<p>LC-MS\/MS analysis of fentanyl and its analogues was performed on an ACQUITY UPLC instrument coupled with a Waters Xevo TQ-S mass spectrometer. Instrument conditions were as follows, and analyte transitions are provided in Table I.<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><tbody><tr><td>Analytical column:<\/td><td colspan=\"2\">Raptor Biphenyl (5 \u00b5m, 50 mm x 2.1 mm;\u00a0cat.# 9309552)<\/td><\/tr><tr><td>Guard column:<\/td><td colspan=\"2\">Raptor Biphenyl EXP guard column cartridge, (5 \u00b5m, 5 mm x 2.1 mm;\u00a0cat.#\u00a0930950252)<\/td><\/tr><tr><td>Mobile phase A:<\/td><td colspan=\"2\">0.1% Formic acid in water<\/td><\/tr><tr><td>Mobile phase B:<\/td><td colspan=\"2\">0.1% Formic acid in methanol<\/td><\/tr><tr><td>Gradient<\/td><td>Time (min)<\/td><td>%B<\/td><\/tr><tr><td>&nbsp;<\/td><td>0.00<\/td><td>30<\/td><\/tr><tr><td>&nbsp;<\/td><td>2.50<\/td><td>70<\/td><\/tr><tr><td>&nbsp;<\/td><td>2.51<\/td><td>30<\/td><\/tr><tr><td>&nbsp;<\/td><td>3.50<\/td><td>30<\/td><\/tr><tr><td>Flow rate:<\/td><td colspan=\"2\">0.4 mL\/min<\/td><\/tr><tr><td>Injection volume:<\/td><td colspan=\"2\">5 \u00b5L<\/td><\/tr><tr><td>Column temp.:<\/td><td colspan=\"2\">40 \u00b0C<\/td><\/tr><tr><td>Ion mode:<\/td><td colspan=\"2\">Positive ESI<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<p><strong>Table I:<\/strong>&nbsp;Analyte Transitions<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><thead><tr><th><strong>Analyte<\/strong><\/th><th><strong>Precursor Ion<\/strong><\/th><th><strong>Product Ion<br>Qualifier<\/strong><\/th><th><strong>Product Ion<br>Qualifier<\/strong><\/th><th><strong>Internal Standard<\/strong><\/th><\/tr><\/thead><tbody><tr><td>Norfentanyl<\/td><td>233.27<\/td><td>84.15<\/td><td>56.06<\/td><td>Norfentanyl-D<sub>5<\/sub><\/td><\/tr><tr><td>Acetyl fentanyl<\/td><td>323.37<\/td><td>188.25<\/td><td>105.15<\/td><td>Acetyl fentanyl-<sup>13<\/sup>C<sub>6<\/sub><\/td><\/tr><tr><td>Fentanyl<\/td><td>337.37<\/td><td>188.26<\/td><td>105.08<\/td><td>Fentanyl-D<sub>5<\/sub><\/td><\/tr><tr><td>Butyryl fentanyl<\/td><td>351.43<\/td><td>188.20<\/td><td>105.15<\/td><td>Carfentanil-D<sub>5<\/sub><\/td><\/tr><tr><td>Remifentanil<\/td><td>377.37<\/td><td>113.15<\/td><td>317.30<\/td><td>Norfentanyl-D<sub>5<\/sub><\/td><\/tr><tr><td>Sufentanil<\/td><td>387.40<\/td><td>238.19<\/td><td>111.06<\/td><td>Sufentanil-D<sub>5<\/sub><\/td><\/tr><tr><td>Carfentanil<\/td><td>395.40<\/td><td>113.14<\/td><td>335.35<\/td><td>Carfentanil-D<sub>5<\/sub><\/td><\/tr><tr><td>Alfentanil<\/td><td>417.47<\/td><td>268.31<\/td><td>197.23<\/td><td>Acetyl fentanyl-<sup>13<\/sup>C<sub>6<\/sub><\/td><\/tr><tr><td>Norfentanyl-D<sub>5<\/sub><\/td><td>238.30<\/td><td>84.15<\/td><td>\u2014<\/td><td>\u2014<\/td><\/tr><tr><td>Acetyl fentanyl-<sup>13<\/sup>C<sub>6<\/sub><\/td><td>329.37<\/td><td>188.25<\/td><td>\u2014<\/td><td>\u2014<\/td><\/tr><tr><td>Fentanyl-D<sub>5<\/sub><\/td><td>342.47<\/td><td>188.27<\/td><td>\u2014<\/td><td>\u2014<\/td><\/tr><tr><td>Sufentanil-D<sub>5<\/sub><\/td><td>392.40<\/td><td>238.25<\/td><td>\u2014<\/td><td>\u2014<\/td><\/tr><tr><td>Carfentanil-D<sub>5<\/sub><\/td><td>400.40<\/td><td>340.41<\/td><td>\u2014<\/td><td>\u2014<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<div style=\"height:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Results and Discussion<\/h2>\n\n\n\n<h3 class=\"wp-block-heading\">Chromatographic Performance<\/h3>\n\n\n\n<p>All eight analytes were well separated within a 2.5-minute gradient elution (3.5-minute total analysis time) on a Raptor Biphenyl column (Figure 2). No significant matrix interference was observed to negatively affect quantification of the five-fold diluted urine samples. The 5 \u00b5m particle Raptor Biphenyl column used here is a superficially porous particle (SPP) column. It was selected for this method in part because it provides similar performance to a smaller particle size fully porous particle (FPP) column, but it generates less system backpressure.<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 2:<\/strong>\u00a0The Raptor Biphenyl column effectively separated all target compounds in urine with no observed matrix interferences.<\/div><div class='chromatogram-article-inner-full'><div class=\"chromatogram-article-inner\">\n\n<p class=\"has-medium-font-size\"><strong>Blank Urine<\/strong><\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article\"><div class=\"wp-block-custom-chromatogram-article\"><div class=\"chromatogram-image wide-image\"><img decoding=\"async\" src=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0716.png\" alt=\"Blank Urine for Comparison to Fentanyl and Analogues on Raptor Biphenyl by LC-MS\/MS\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0716<\/p><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 Biphenyl  (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9309552?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0716\" rel=\"noopener\">cat.# 9309552<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Dimensions:<\/th><td>50 mm x 2.1 mm ID<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Particle Size:<\/th><td>5 \u00b5m<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Pore Size:<\/th><td>90 \u00c5<\/td><\/tr><tr><td><\/td><tr><th class=\"sub conditions_header\" scope=\"row\">Guard Column:<\/th><td>Raptor Biphenyl EXP guard cartridge 5 mm, 2.1 mm ID, 5 \u00b5m (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/930950252?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0716\" rel=\"noopener\">cat.# 930950252<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Temp.:<\/th><td>40 \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>70:30 Water:methanol<\/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>0.1% Formic acid in water <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">B:<\/th><td>0.1% Formic acid in methanol <\/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>0.4<\/td><td>70<\/td><td>30<\/td><\/tr><tr><td>2.50<\/td><td>0.4<\/td><td>30<\/td><td>70<\/td><\/tr><tr><td>2.51<\/td><td>0.4<\/td><td>70<\/td><td>30<\/td><\/tr><tr><td>3.50<\/td><td>0.4<\/td><td>70<\/td><td>30<\/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>An 80 \u03bcL aliquot of blank urine was mixed with 320 \u03bcL of 70:30 water:methanol solution (5-fold dilution) and 10 \u03bcL of internal standard solution (40 ng\/mL in methanol) in a Thomson SINGLE StEP filter vial (Restek cat.# 25895). After filtering through the 0.2 \u00b5m PVDF membrane, 5 \u03bcL was injected for analysis. <\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-related-skus\"><h4>Related SKUs<\/h4><ul><li>930950252<\/li><li>9309552<\/li><\/ul><\/div><div class=\"chromatogram-related-searches\"><h4>Related Searches<\/h4><ul><li>LC-MS\/MS<\/li><li>Raptor<\/li><li>acetyl fentanyl<\/li><li>alfentanil<\/li><li>biphenyl<\/li><li>butyryl fentanyl<\/li><li>carfentanil<\/li><li>fentanyl<\/li><li>norfentanyl<\/li><li>remifentanil<\/li><li>sufentani<\/li><\/ul><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0716.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\n\n<p class=\"has-medium-font-size\"><strong>10 ng\/mL Urine Standard<\/strong><\/p>\n\n\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_cf0715.png\" alt=\"Fentanyl and Analogues in Urine on Raptor Biphenyl by LC-MS\/MS\" title=\"-\"><\/div><p class=\"article-id\" style=\"text-align:center\"> LC_CF0715<\/p><div class=\"chromatogram-peaks\"><h4>Peaks<\/h4><table class=\"peaks col-12\">\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\">Conc.<br \/>(ng\/mL)<\/th><th style=\"text-align: center;width: 75px\">Precursor Ion<\/th><th style=\"text-align: center;width: 75px\">Product 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\">Norfentanyl-d5 <\/td><td class=\"oth\">1.03<\/td><td class=\"oth\">1<\/td><td class=\"oth\">238.30<\/td><td class=\"oth\">84.15<\/td><td class=\"oth\">&#8211;<\/td><\/tr>\n<tr><td class=\"num\">2.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Norfentanyl\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/1609-66-1\/Norfentanyl\" target=\"_blank\" rel=\"noopener\">Norfentanyl<\/a><\/td><td class=\"oth\">1.04<\/td><td class=\"oth\">10<\/td><td class=\"oth\">233.27<\/td><td class=\"oth\">84.15<\/td><td class=\"oth\">56.06<\/td><\/tr>\n<tr><td class=\"num\">3.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Remifentanil\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/132875-61-7\/Remifentanil\" target=\"_blank\" rel=\"noopener\">Remifentanil<\/a><\/td><td class=\"oth\">1.34<\/td><td class=\"oth\">10<\/td><td class=\"oth\">377.37<\/td><td class=\"oth\">113.15<\/td><td class=\"oth\">317.30<\/td><\/tr>\n<tr><td class=\"num\">4.<\/td><td class=\"cmpd\">Acetyl fentanyl-13C6<\/td><td class=\"oth\">1.69<\/td><td class=\"oth\">1<\/td><td class=\"oth\">329.37<\/td><td class=\"oth\">188.25<\/td><td class=\"oth\">&#8211;<\/td><\/tr>\n<tr><td class=\"num\">5.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Acetyl fentanyl\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/3258-84-2\/Acetyl fentanyl\" target=\"_blank\" rel=\"noopener\">Acetyl fentanyl<\/a><\/td><td class=\"oth\">1.69<\/td><td class=\"oth\">10<\/td><td class=\"oth\">323.37<\/td><td class=\"oth\">188.25<\/td><td class=\"oth\">105.15<\/td><\/tr>\n<tr><td class=\"num\">6.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Alfentanil\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/71195-58-9\/Alfentanil\" target=\"_blank\" rel=\"noopener\">Alfentanil<\/a><\/td><td class=\"oth\">1.88<\/td><td class=\"oth\">10<\/td><td class=\"oth\">417.47<\/td><td class=\"oth\">268.31<\/td><td class=\"oth\">197.23<\/td><\/tr>\n<tr><td class=\"num\">7.<\/td><td class=\"cmpd\">Fentanyl-d5<\/td><td class=\"oth\">1.95<\/td><td class=\"oth\">1<\/td><td class=\"oth\">342.47<\/td><td class=\"oth\">188.27<\/td><td class=\"oth\">&#8211;<\/td><\/tr>\n<tr><td class=\"num\">8.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Fentanyl\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/437-38-7\/Fentanyl\" target=\"_blank\" rel=\"noopener\">Fentanyl<\/a><\/td><td class=\"oth\">1.96<\/td><td class=\"oth\">10<\/td><td class=\"oth\">337.37<\/td><td class=\"oth\">188.26<\/td><td class=\"oth\">105.08<\/td><\/tr>\n<tr><td class=\"num\">9.<\/td><td class=\"cmpd\">Carfentanil-d5<\/td><td class=\"oth\">2.04<\/td><td class=\"oth\">1<\/td><td class=\"oth\">400.40<\/td><td class=\"oth\">340.41<\/td><td class=\"oth\">&#8211;<\/td><\/tr>\n<tr><td class=\"num\">10.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Carfentanil\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/59708-52-0\/Carfentanil\" target=\"_blank\" rel=\"noopener\">Carfentanil<\/a><\/td><td class=\"oth\">2.05<\/td><td class=\"oth\">10<\/td><td class=\"oth\">395.40<\/td><td class=\"oth\">113.14<\/td><td class=\"oth\">335.35<\/td><\/tr>\n<tr><td class=\"num\">11.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Butyryl fentanyl\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/1443-52-3\/Butyryl fentanyl\" target=\"_blank\" rel=\"noopener\">Butyryl fentanyl<\/a><\/td><td class=\"oth\">2.15<\/td><td class=\"oth\">10<\/td><td class=\"oth\">351.43<\/td><td class=\"oth\">188.20<\/td><td class=\"oth\">105.15<\/td><\/tr>\n<tr><td class=\"num\">12.<\/td><td class=\"cmpd\">Sufentanil-d5<\/td><td class=\"oth\">2.25<\/td><td class=\"oth\">1<\/td><td class=\"oth\">392.40<\/td><td class=\"oth\">238.25<\/td><td class=\"oth\">&#8211;<\/td><\/tr>\n<tr><td class=\"num\">13.<\/td><td class=\"cmpd\"><a class=\"cmpd_link\" title=\"View compound information for Sufentanil\" href=\"https:\/\/ez.restek.com\/compound\/view\/en\/56030-54-7\/Sufentanil\" target=\"_blank\" rel=\"noopener\">Sufentanil<\/a><\/td><td class=\"oth\">2.26<\/td><td class=\"oth\">10<\/td><td class=\"oth\">387.40<\/td><td class=\"oth\">238.19<\/td><td class=\"oth\">111.06<\/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 Biphenyl  (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/9309552?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0715\" rel=\"noopener\">cat.# 9309552<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Dimensions:<\/th><td>50 mm x 2.1 mm ID<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Particle Size:<\/th><td>5 \u00b5m<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Pore Size:<\/th><td>90 \u00c5<\/td><\/tr><tr><td><\/td><tr><th class=\"sub conditions_header\" scope=\"row\">Guard Column:<\/th><td>Raptor Biphenyl EXP guard cartridge 5 mm, 2.1 mm ID, 5 \u00b5m (<a target=\"_blank\" href=\"https:\/\/www.restek.com\/p\/930950252?utm_source=chromatograms&amp;utm_medium=link&amp;utm_campaign=LC_CF0715\" rel=\"noopener\">cat.# 930950252<\/a>)<\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">Temp.:<\/th><td>40 \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>70:30 Water:methanol<\/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>0.1% Formic acid in water <\/td><\/tr><tr><th class=\"sub conditions_header\" scope=\"row\">B:<\/th><td>0.1% Formic acid in methanol <\/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>0.4<\/td><td>70<\/td><td>30<\/td><\/tr><tr><td>2.50<\/td><td>0.4<\/td><td>30<\/td><td>70<\/td><\/tr><tr><td>2.51<\/td><td>0.4<\/td><td>70<\/td><td>30<\/td><\/tr><tr><td>3.50<\/td><td>0.4<\/td><td>70<\/td><td>30<\/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>Human urine was fortified at 10 ng\/mL with target analytes. An 80 \u03bcL urine aliquot was mixed with 320 \u03bcL of 70:30 water:methanol solution (5-fold dilution) and 10 \u03bcL of internal standard solution (40 ng\/mL in methanol) in a Thomson SINGLE StEP filter vial (Restek cat.# 25895). After filtering through the 0.2 \u00b5m PVDF membrane, 5 \u03bcL was injected for analysis. <\/td><\/tr><\/table><\/div><\/div><\/div><div class=\"chromatogram-pdf-link\"><a href=\"https:\/\/ez.restek.com\/images\/cgram\/lc_cf0715.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:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\">Linearity<\/h3>\n\n\n\n<p>Linear responses were obtained for all compounds and the calibration ranges encompassed typical concentration levels monitored for both research and abuse. Using 1\/x weighted linear regression (1\/x<sup>2<\/sup>&nbsp;for butyryl fentanyl), calibration linearity ranged from 0.05 to 50 ng\/mL for fentanyl, alfentanil, acetyl fentanyl, butyryl fentanyl, and sufentanil; from 0.10 to 50 ng\/mL for remifentanil; and from 0.25 to 50 ng\/mL for norfentanyl and carfentanil. All analytes showed acceptable linearity with r<sup>2<\/sup>&nbsp;values of 0.996 or greater (Figure 3) and deviations of &lt;12% (&lt;20% for the lowest concentrated standard).<\/p>\n\n\n<div class=\"wp-block-custom-chromatogram-article-top\"><div class=\"chromatogram-article-placeholder\"><div class=\"figure-heading\"><strong>Figure 3:<\/strong>\u00a0Calibration Curves<\/div><div class='chromatogram-article-inner-full'><div class=\"chromatogram-article-inner\">\n<style>.kb-image43348_882a74-88 .kb-image-has-overlay:after{opacity:0.3;}<\/style>\n<div class=\"wp-block-kadence-image kb-image43348_882a74-88\"><figure class=\"aligncenter size-large\"><img loading=\"lazy\" decoding=\"async\" width=\"783\" height=\"1024\" src=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN2820-03-783x1024.jpg\" alt=\"\" class=\"kb-img wp-image-18934\" title=\"-\" srcset=\"https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN2820-03-783x1024.jpg 783w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN2820-03-229x300.jpg 229w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN2820-03-768x1004.jpg 768w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN2820-03-1175x1536.jpg 1175w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN2820-03-1566x2048.jpg 1566w, https:\/\/discover.restek.com\/wp-content\/uploads\/figure-article-CFAN2820-03.jpg 1800w\" sizes=\"auto, (max-width: 783px) 100vw, 783px\" \/><\/figure><\/div>\n\n<\/div><\/div><\/div>\n\n\n<div style=\"height:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h3 class=\"wp-block-heading\">Accuracy and Precision<\/h3>\n\n\n\n<p>Based on three independent experiments conducted on multiple days, method accuracy for the analysis of fentanyl and its analogues was demonstrated by the %recovery values, which were within 10% of the nominal concentration for all compounds at all QC levels. The %RSD range was 0.5-8.3% and 3.4-8.4% for intraday and interday comparisons, respectively, indicating acceptable method precision (Table II).<\/p>\n\n\n\n<p><strong>Table II:<\/strong>&nbsp;Accuracy and Precision Results for Fentanyl and Related Compounds in Urine QC Samples.<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table class=\"has-fixed-layout\"><tbody><tr><td>&nbsp;<\/td><td colspan=\"3\"><strong>QC Level 1 (0.750 ng\/mL)<\/strong><\/td><td colspan=\"3\"><strong>QC Level 2 (4.00 ng\/mL)<\/strong><\/td><td colspan=\"3\"><strong>QC Level 3 (20.0 ng\/mL)<\/strong><\/td><\/tr><tr><td><strong>Analyte<\/strong><\/td><td><strong>Average Conc. (ng\/mL)<\/strong><\/td><td><strong>Average Accuracy (%)<\/strong><\/td><td><strong>%RSD<\/strong><\/td><td><strong>Average Conc. (ng\/mL)<\/strong><\/td><td><strong>Average Accuracy (%)<\/strong><\/td><td><strong>%RSD<\/strong><\/td><td><strong>Average Conc. (ng\/mL)<\/strong><\/td><td><strong>Average Accuracy (%)<\/strong><\/td><td><strong>%RSD<\/strong><\/td><\/tr><tr><td>Acetyl fentanyl<\/td><td>0.761<\/td><td>102<\/td><td>1.54<\/td><td>3.99<\/td><td>99.7<\/td><td>2.08<\/td><td>19.9<\/td><td>99.3<\/td><td>0.856<\/td><\/tr><tr><td>Alfentanil<\/td><td>0.733<\/td><td>97.6<\/td><td>3.34<\/td><td>3.96<\/td><td>98.9<\/td><td>8.38<\/td><td>20.9<\/td><td>104<\/td><td>6.73<\/td><\/tr><tr><td>Butyryl fentanyl<\/td><td>0.741<\/td><td>98.9<\/td><td>6.29<\/td><td>3.77<\/td><td>94.3<\/td><td>6.01<\/td><td>20.8<\/td><td>104<\/td><td>4.95<\/td><\/tr><tr><td>Carfentanil<\/td><td>0.757<\/td><td>101<\/td><td>7.34<\/td><td>3.76<\/td><td>94.0<\/td><td>4.64<\/td><td>20.6<\/td><td>103<\/td><td>4.24<\/td><\/tr><tr><td>Fentanyl<\/td><td>0.761<\/td><td>102<\/td><td>1.98<\/td><td>3.96<\/td><td>99.1<\/td><td>2.31<\/td><td>19.9<\/td><td>99.6<\/td><td>1.04<\/td><\/tr><tr><td>Norfentanyl<\/td><td>0.768<\/td><td>103<\/td><td>6.50<\/td><td>4.04<\/td><td>101<\/td><td>1.84<\/td><td>20.1<\/td><td>101<\/td><td>2.55<\/td><\/tr><tr><td>Remifentanil<\/td><td>0.765<\/td><td>102<\/td><td>3.42<\/td><td>3.97<\/td><td>99.2<\/td><td>3.68<\/td><td>20.8<\/td><td>104<\/td><td>4.14<\/td><\/tr><tr><td>Sufentanil<\/td><td>0.752<\/td><td>100<\/td><td>1.67<\/td><td>3.93<\/td><td>98.3<\/td><td>1.28<\/td><td>20.1<\/td><td>100<\/td><td>0.943<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<div style=\"height:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Conclusion<\/h2>\n\n\n\n<p>A simple dilute-and-shoot method was developed for the quantitative analysis of fentanyl and its analogues in human urine. The analytical method was demonstrated to be fast, rugged, and sensitive with acceptable accuracy and precision for urine sample analysis. The Raptor Biphenyl column is well suited for the analysis of these synthetic opioid compounds and this method can be applied to clinical toxicology, forensic analysis, workplace drug testing, and pharmaceutical research.<\/p>\n\n\n\n<div style=\"height:30px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n\n\n        <div class=\"cpb\">\n            <h3 class=\"cpb-heading\">Products Mentioned<\/h3>\n            <hr class=\"cpb-heading-underline\" \/>\n            <div class=\"cpb-list\">\n                                    <div class=\"cpb-item\">\n                        <div class=\"cpb-col cpb-col--left\">\n                            <a class=\"cpb-catalog\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/9309552\">                                Catalog No. 9309552                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Raptor Biphenyl, 5 \u00b5m, 50 x 2.1 mm HPLC\u30ab\u30e9\u30e0<\/div>\n                        <\/div>\n                        <div class=\"cpb-col cpb-col--right\">\n                                                            <a class=\"cpb-view-btn\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/9309552\">\u88fd\u54c1\u60c5\u5831\u3092\u898b\u308b<\/a>\n                                                    <\/div>\n                    <\/div>\n                                    <div class=\"cpb-item\">\n                        <div class=\"cpb-col cpb-col--left\">\n                            <a class=\"cpb-catalog\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/930950252\">                                Catalog No. 930950252                            <\/a>                        <\/div>\n                        <div class=\"cpb-col cpb-col--middle\">\n                            <div class=\"cpb-title\">Raptor Biphenyl EXP Guard Column Cartridge, 5 \u00b5m, 5 x 2.1 mm, 3-pk.<\/div>\n                        <\/div>\n                        <div class=\"cpb-col cpb-col--right\">\n                                                            <a class=\"cpb-view-btn\" target=\"_blank\" rel=\"noopener noreferrer\" href=\"https:\/\/www.restek.com\/p\/930950252\">\u88fd\u54c1\u60c5\u5831\u3092\u898b\u308b<\/a>\n                                                    <\/div>\n                    <\/div>\n                            <\/div>\n        <\/div>\n        \n\n\n<div style=\"height:100px\" aria-hidden=\"true\" class=\"wp-block-spacer\"><\/div>\n","protected":false},"excerpt":{"rendered":"<p>Synthetic opioid drugs, such as fentanyl and sufentanil, have very high analgesic potency. Abuse of these prescription painkillers\u2014along with a rapidly growing list of illicit analogues\u2014is a significant public health problem. In this study, we developed a simple dilute-and-shoot method that provides a fast 3.5 minute analysis of fentanyl and related compounds (norfentanyl, acetyl fentanyl, alfentanil, butyryl fentanyl, carfentanil, remifentanil, and sufentanil) in human urine by LC-MS\/MS using a Raptor Biphenyl column.<\/p>\n","protected":false},"author":46,"featured_media":6349,"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":[13],"tags":[],"industries-application":[2153,2156,2170,2171,2172],"post-badge":[],"resource-type":[],"product-library":[2391,2373,2375,2402,2405],"resource-technique":[2299,2302],"ppma_author":[578,586],"class_list":["post-43348","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-application-notes","industries-application-clinical","industries-application-clinical-toxicology","industries-application-forensics","industries-application-forensics-toxicology","industries-application-pharmaceutical-biopharma","product-library-lc-columns","product-library-liquid-chromatography-products","product-library-sample-preparation-products","product-library-spe-sle","product-library-vials-accessories","resource-technique-liquid-chromatography","resource-technique-ms-ms"],"acf":[],"taxonomy_info":{"category":[{"value":13,"label":"Application Notes"}],"industries-application":[{"value":2153,"label":"Clinical"},{"value":2156,"label":"Clinical Toxicology"},{"value":2170,"label":"Forensics"},{"value":2171,"label":"Forensics Toxicology"},{"value":2172,"label":"Pharmaceutical &amp; Biopharma"}],"product-library":[{"value":2391,"label":"LC Columns"},{"value":2373,"label":"Liquid Chromatography Products"},{"value":2375,"label":"Sample Preparation Products"},{"value":2402,"label":"SPE &amp; SLE"},{"value":2405,"label":"Vials &amp; Accessories"}],"resource-technique":[{"value":2299,"label":"Liquid Chromatography"},{"value":2302,"label":"MS\/MS"}]},"featured_image_src_large":["https:\/\/discover.restek.com\/wp-content\/uploads\/feature-CFAN2820-1024x536.jpg",1024,536,true],"author_info":{"display_name":"Shun-Hsin Liang, PhD","author_link":"https:\/\/discover.restek.com\/ja\/author\/shun-hsin-liang-phd\/"},"comment_info":0,"category_info":[{"term_id":13,"name":"Application Notes","slug":"application-notes","term_group":0,"term_taxonomy_id":13,"taxonomy":"category","description":"","parent":0,"count":87,"filter":"raw","cat_ID":13,"category_count":87,"category_description":"","cat_name":"Application Notes","category_nicename":"application-notes","category_parent":0}],"tag_info":false,"authors":[{"term_id":578,"user_id":46,"is_guest":0,"slug":"shun-hsin-liang-phd","display_name":"Shun-Hsin Liang, PhD","avatar_url":{"url":"https:\/\/discover.restek.com\/wp-content\/uploads\/people-liang-shun-hsin-e1765414149534.png","url2x":"https:\/\/discover.restek.com\/wp-content\/uploads\/people-liang-shun-hsin-e1765414149534.png"},"0":null,"1":"","2":"","3":"","4":"","5":"","6":"","7":"","8":""},{"term_id":586,"user_id":0,"is_guest":1,"slug":"frances-carroll","display_name":"Frances Carroll","avatar_url":"https:\/\/secure.gravatar.com\/avatar\/?s=96&d=mm&r=g","0":null,"1":"","2":"","3":"","4":"","5":"","6":"","7":"","8":""}],"_links":{"self":[{"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/posts\/43348","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/users\/46"}],"replies":[{"embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/comments?post=43348"}],"version-history":[{"count":9,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/posts\/43348\/revisions"}],"predecessor-version":[{"id":85541,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/posts\/43348\/revisions\/85541"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/media\/6349"}],"wp:attachment":[{"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/media?parent=43348"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/categories?post=43348"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/tags?post=43348"},{"taxonomy":"industries-application","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/industries-application?post=43348"},{"taxonomy":"post-badge","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/post-badge?post=43348"},{"taxonomy":"resource-type","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/resource-type?post=43348"},{"taxonomy":"product-library","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/product-library?post=43348"},{"taxonomy":"resource-technique","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/resource-technique?post=43348"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/discover.restek.com\/ja\/wp-json\/wp\/v2\/ppma_author?post=43348"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}