Optimization and Validation of a High Throughput UHPLC-MS/MS Method for Determination of the EU Regulated Lipophilic Marine Toxins and Occurrence in Fresh and Processed Shellfish
Under the name of lipophilic marine toxins, there are included more than 1000 toxic secondary metabolites, produced by phytoplankton, with the common chemical property of lipophilicity. Due to toxicological effects and geographical distribution, in European legislation relevant compounds are regulat...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-02-01
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| Series: | Marine Drugs |
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| Online Access: | https://www.mdpi.com/1660-3397/20/3/173 |
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| author | Teresa D’Amore Sonia Lo Magro Valeria Vita Aurelia Di Taranto |
| author_facet | Teresa D’Amore Sonia Lo Magro Valeria Vita Aurelia Di Taranto |
| author_sort | Teresa D’Amore |
| collection | DOAJ |
| description | Under the name of lipophilic marine toxins, there are included more than 1000 toxic secondary metabolites, produced by phytoplankton, with the common chemical property of lipophilicity. Due to toxicological effects and geographical distribution, in European legislation relevant compounds are regulated, and their determination is accomplished with the reference liquid chromatography-tandem mass spectrometry method. In this study a modified ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed for the identification and quantification of EU-regulated lipophilic toxins. The method optimization included a refinement of SPE-C18 clean-up, in order to reduce matrix interferences. Improved LC conditions and upgraded chromatographic ammonia-based gradient ensured the best separation of all analytes and, in particular, of the two structural isomers (OA and DTX2). Also, different MS parameters were tested, and confirmation criteria finally established. The validation studies confirmed that all parameters were satisfactory. The requirements for precision (RSD% < 11.8% for each compound), trueness (recoveries from 73 to 101%) and sensitivity (limits of quantification in the range 3–8 µg kg<sup>−1</sup>) were fulfilled. The matrix effect, ranging from −9 to 19%, allowed the use of a calibration curve in solvent (3–320 µg kg<sup>−1</sup> in matrix) for quantification of real samples. Method relative uncertainty ranged from 12 to 20.3%. Additionally, a total of 1000 shellfish samples was analysed, providing a first preliminary surveillance study that may contribute to the knowledge of lipophilic marine toxins contamination. Increase in algae proliferation events and intoxication cases, EFSA suggestions for modification of maximum permitted levels and toxicity equivalency factors, and new studies of important toxic effects underline that implementation of reference methods still represents an important task for health and food safety laboratories. |
| first_indexed | 2024-03-09T13:30:54Z |
| format | Article |
| id | doaj.art-22ba4decae894303b6965964e51696df |
| institution | Directory Open Access Journal |
| issn | 1660-3397 |
| language | English |
| last_indexed | 2024-03-09T13:30:54Z |
| publishDate | 2022-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Marine Drugs |
| spelling | doaj.art-22ba4decae894303b6965964e51696df2023-11-30T21:17:50ZengMDPI AGMarine Drugs1660-33972022-02-0120317310.3390/md20030173Optimization and Validation of a High Throughput UHPLC-MS/MS Method for Determination of the EU Regulated Lipophilic Marine Toxins and Occurrence in Fresh and Processed ShellfishTeresa D’Amore0Sonia Lo Magro1Valeria Vita2Aurelia Di Taranto3Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia 20, 71121 Foggia, ItalyIstituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia 20, 71121 Foggia, ItalyIstituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia 20, 71121 Foggia, ItalyIstituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Via Manfredonia 20, 71121 Foggia, ItalyUnder the name of lipophilic marine toxins, there are included more than 1000 toxic secondary metabolites, produced by phytoplankton, with the common chemical property of lipophilicity. Due to toxicological effects and geographical distribution, in European legislation relevant compounds are regulated, and their determination is accomplished with the reference liquid chromatography-tandem mass spectrometry method. In this study a modified ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed for the identification and quantification of EU-regulated lipophilic toxins. The method optimization included a refinement of SPE-C18 clean-up, in order to reduce matrix interferences. Improved LC conditions and upgraded chromatographic ammonia-based gradient ensured the best separation of all analytes and, in particular, of the two structural isomers (OA and DTX2). Also, different MS parameters were tested, and confirmation criteria finally established. The validation studies confirmed that all parameters were satisfactory. The requirements for precision (RSD% < 11.8% for each compound), trueness (recoveries from 73 to 101%) and sensitivity (limits of quantification in the range 3–8 µg kg<sup>−1</sup>) were fulfilled. The matrix effect, ranging from −9 to 19%, allowed the use of a calibration curve in solvent (3–320 µg kg<sup>−1</sup> in matrix) for quantification of real samples. Method relative uncertainty ranged from 12 to 20.3%. Additionally, a total of 1000 shellfish samples was analysed, providing a first preliminary surveillance study that may contribute to the knowledge of lipophilic marine toxins contamination. Increase in algae proliferation events and intoxication cases, EFSA suggestions for modification of maximum permitted levels and toxicity equivalency factors, and new studies of important toxic effects underline that implementation of reference methods still represents an important task for health and food safety laboratories.https://www.mdpi.com/1660-3397/20/3/173biotoxinsokadaic acidyessotoxinpectenotoxinazaspiracidUHPLC-MS/MS |
| spellingShingle | Teresa D’Amore Sonia Lo Magro Valeria Vita Aurelia Di Taranto Optimization and Validation of a High Throughput UHPLC-MS/MS Method for Determination of the EU Regulated Lipophilic Marine Toxins and Occurrence in Fresh and Processed Shellfish Marine Drugs biotoxins okadaic acid yessotoxin pectenotoxin azaspiracid UHPLC-MS/MS |
| title | Optimization and Validation of a High Throughput UHPLC-MS/MS Method for Determination of the EU Regulated Lipophilic Marine Toxins and Occurrence in Fresh and Processed Shellfish |
| title_full | Optimization and Validation of a High Throughput UHPLC-MS/MS Method for Determination of the EU Regulated Lipophilic Marine Toxins and Occurrence in Fresh and Processed Shellfish |
| title_fullStr | Optimization and Validation of a High Throughput UHPLC-MS/MS Method for Determination of the EU Regulated Lipophilic Marine Toxins and Occurrence in Fresh and Processed Shellfish |
| title_full_unstemmed | Optimization and Validation of a High Throughput UHPLC-MS/MS Method for Determination of the EU Regulated Lipophilic Marine Toxins and Occurrence in Fresh and Processed Shellfish |
| title_short | Optimization and Validation of a High Throughput UHPLC-MS/MS Method for Determination of the EU Regulated Lipophilic Marine Toxins and Occurrence in Fresh and Processed Shellfish |
| title_sort | optimization and validation of a high throughput uhplc ms ms method for determination of the eu regulated lipophilic marine toxins and occurrence in fresh and processed shellfish |
| topic | biotoxins okadaic acid yessotoxin pectenotoxin azaspiracid UHPLC-MS/MS |
| url | https://www.mdpi.com/1660-3397/20/3/173 |
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