Quantification of Linear Alkylbenzene Sulphonates in Complex Sludge Samples: Influence of Matrix Effects in Calibration Methods

Mic ochemical Jou nal 204 (2024) 111089 A ailable online 28 June 2024 0026-265X/© 2024 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by- nc-nd/4.0/). Quan i ica ion o linea alkylbenzene sulphona es in complex sludge samples: In luence o ma ix e ec s in calib a ion me hods Julia Ma ín a , * , Ca men Mejías a , Juan Luis San os a , I ene Apa icio a , Es eban Alonso a , John Heinze b a Depa amen o de Química Analí ica, Escuela Poli ´ ecnica Supe io , Uni e sidad de Se illa. E-41011 Se ille, Spain b Council o LAB/LAS En i onmen al Resea ch (CLER), 529 14 h S ee , NW, Sui e 1280, Washing on, DC 20045, USA ARTICLE INFO Keywo ds: Linea alkylbenzene sulphona e Calib a ion me hods Ma ix e ec Sludge samples ABSTRACT Linea Alkylbenzene Sul ona es (LAS) a e among he p io i y o ganic pollu an s in sludge owing o hei high concen a ions, due o i s use as a majo cleaning agen (su ac an ) in laund y de e gen s and household cleaning p oduc s, and o he concen a ion limi s o 2,600 mg/kg ou lined in he EU di ec i e d a o land applica ion o sludge. In his wo k, a me hod has been op imized and alida ed o he accu a e quan i ica ion o LAS homologues C10-C13 in di e en ypes o samples in he sludge ea men and disposal p ocess including p ima y, seconda y, and diges ed sludge, compos , and soil. The ype o ma ix had a signi ican impac on he me hod’s alida ion p ocess, esul ing in he need o he use o dis inc dilu ion ac o s o each ma ix. O e all, he p ocedu e p opose in ol es a cos -e ec i e and use - iendly echnique ha combines bo h ex ac ion and clean-up in a single s ep (ul asound-assis ed ex ac ion and dispe si e solid phase ex ac ion). Analy ical de e mina ion is conduc ed using liquid ch oma og aphy- andem mass spec ome y. The me hod demons a ed excellen accu acy ( ela i e eco e ies exceeding 84.5 % ac oss all ypes o sludge samples) and he limi o quan i ica ion enables he measu emen o LAS concen a ions up o 1000 imes lowe han he concen a ion limi s s ipula ed in he EU di ec i e d a . Fo an accu a e quan i ica ion, h ee calib a ion app oaches we e es ed (ex e nal calib a ion, s anda d addi ion, and ma ix-ma ched calib a ion). S anda d addi ion was he p e e ed me hod o quan i a i e analysis. I helps mi iga e ma ix e ec s ha could o he wise dis o he ac- cu acy o analy e measu emen s, making i c ucial o quali y con ol, especially in moni o ing applica ions whe e ma ix e ec s may in oduce bias in o concen a ion calcula ions. 1. In oduc ion Wi hin he Eu opean Union (EU), he ypical and p e alen p ac ice o sewage sludge disposal in ol es i s u iliza ion as an o ganic soil amendmen . This choice is no only cos -e ec i e bu also en i on- men ally bene icial, as i p o ides a aluable sou ce o o ganic ma e , ni ogen, and phospho ous, making i a use ul e ilize and soil condi- ione [1]. Howe e , his p ac ice comes wi h po en ial isks due o he p esence o hea y me als and o ganic pollu an s a high concen a ion le els in sludge. These con aminan s end o accumula e in sludge and, subsequen ly, can ind hei way in o he soil [2]. Fo ins ance, Gonzalez e al. (2012) [3] ound concen a ions o Linea Alkylbenzene Sul ona es (LAS) in compos -amended soil up o 18.7 mg/kg d y weigh (dw). LAS a e a majo cleaning agen (su ac an ) used in laund y de e gen s and household cleaning p oduc s. The EU Di ec i e 86/278/EEC [4] has egula ed he use in ag icul- u e o esidual sludge om domes ic and u ban was ewa e bu does no egula e he concen a ion o o ganic pollu an s. In 2000, o add ess he cu en challenges in sludge managemen and mi iga e he p esence o pollu an s in soils, he EU in oduced he hi d d a o a u u e sludge di ec i e i led “Wo king Documen on Sludge” [1]. This d a ma ked a signi ican s ep as i in oduced concen a ion limi s o se en g oups o o ganic compounds o he i s ime. Among hese o ganic pollu an s, LAS a e no ewo hy because hei concen a ions in anae obically s a- bilized o anae obic diges ed sludges exceeding he EU di ec i e d a limi s (2,600 mg/kg) [5]. Cu en ly, egula ions ega ding LAS limi s in sludge a e no s anda dized ac oss EU membe s a es, Denma k has implemen ed igo ous measu es by es ablishing a LAS limi o 1,300 mg/kg dw, whe eas Belgium has se i s limi sligh ly highe a 1,500 mg/ kg dw [6]. Fu he mo e, he scien i ic communi y has mainly ocused * Co esponding au ho a : Depa amen o de Química Analí ica, Escuela Poli ´ ecnica Supe io , Uni e sidad de Se illa, c/ Vi gen de ´ A ica, 7, 41011 Se ille, Spain. E-mail add ess: [email p o ec ed] (J. Ma ín). Con en s lis s a ailable a ScienceDi ec Mic ochemical Jou nal jou nal homepage: www.else ie .com/loca e/mic oc h ps://doi.o g/10.1016/j.mic oc.2024.111089 Recei ed 7 May 2024; Recei ed in e ised o m 18 June 2024; Accep ed 27 June 2024 Mic ochemical Jou nal 204 (2024) 111089 2 s udy on eme ging pollu an s [7]. Hence, he impe a i e lies in eliable and easy- o-pe o m analy ical me hods o he p ecise quan i ica ion o di e en homologues o such pollu an s in complex sludge samples. Acco ding o Wo ld Heal h O - ganiza ion, he pe cen age dis ibu ion o LAS homologues commonly used in laund y de e gen s in Eu ope is as ollows: C10 (10–15 %), C11 (25–35 %), C12 (25–35 %), C13 (15–30 %) [8]. Analy ical me hodolo- gies de eloped ea lie p edominan ly concen a ed on he mul i esidue de e mina ion o di e en pollu an s, esul ing in minimal endea o s o alida e simple, au oma ed, and en i onmen ally iendly me hods o Table 1 Op imized pa ame e s o he MS/MS de e mina ion a e LC sepa a ion. Compound P ecu so ion (m/z) MRM 1 a MRM 2 b F agmen o (V) Collision ene gy (eV) LAS C10 297 [M−H] - 183 119 180 36/50 LAS C11 311 [M−H] - 183 119 175 36/50 LAS C12 325 [M−H] - 183 119 180 40/50 LAS C13 339 [M−H] - 183 119 180 40/50 a : Quan i a ion ion; b : Con i ma ion ion. Fig. 1. In luence o clean-up so ben on he LAS eco e y (n =3). Fig. 2. In luence o ex ac ion echnique on he LAS eco e y (n =3). Fig. 3. Global esponse su ace plo s, co esponding o he desi abili y unc ion, when op imizing he ollowing pai o ac o s om he ex ac ion p ocedu e: (cycles s. so ben amoun (A), sample amoun s. so ben amoun (B), and cycles s. sample amoun (C)). Resul s we e e alua ed using a 95 % con idence in e al. J. Ma ín e al. Mic ochemical Jou nal 204 (2024) 111089 3 de e mining LAS. Fo liquid ch oma og aphy (LC) de e mina ion, less expensi e ul a iole (UV) de ec ion has been an op ion [3]. Howe e , he highe amoun o noise in he LC-UV sys em a ec s selec i i y, ac- cu acy and sensi i i y and is he eason why i has been widely eplaced by mass spec ome y (MS) analyze s such as andem mass spec ome y (MS/MS). Howe e , elec osp ay ioniza ion can in oduce ma ix e ec s [9,10], pa icula ly when co-elu ing in e e ences a e p esen in he ma ix ex ac along wi h a ge analy es. I is o u mos impo ance o conside hese ma ix e ec s [11], as hey can po en ially al e analy - ical esul s. Se e al s a egies exis o mi iga e ma ix e ec s [12,13], such as sample ex ac dilu ion [14], albei a he cos o inc eased de ec ion limi s, sample ex ac clean-up, o he use o in e nal s anda ds such as 13 C 2 -LAS-C12, which can be cos ly and challenging o acqui e. Ano he app oach o add ess ma ix e ec s in ol es he u iliza ion o a ious calib a ion me hods, including ma ix-ma ched calib a ion cu es o he s anda d addi ion calib a ion me hod [15–17]. The objec i e o his esea ch is o op imize a s aigh o wa d analy ical me hod o he accu a e quan i ica ion o LAS homologues C10-C13 commonly used in laund y de e gen s in Eu ope in complex sludge samples. The sample ea men in ol es a cos -e ec i e and use - iendly echnique ha combines bo h ex ac ion and clean-up in a single s ep (ul asound-assis ed ex ac ion and dispe si e solid phase ex ac ion). Analy ical de e mina ion is conduc ed using LC-MS/MS. The alida ion was pe o med using h ee di e en calib a ion s a e- gies o de e mine he mos sui able app oach. 2. Expe imen al 2.1. Chemicals and eagen s LC-g ade ace oni ile (ACN), me hanol (MeOH) and wa e we e supplied by Romil (Ba celona, Spain). Analy ical-g ade ammonium ac- e a e (≥98 %) was p o ided by Pan eac (Ba celona, Spain). P ima y- seconda y amine (PSA), C18 and o mic acid we e p o ided by Scha - lab (Ba celona, Spain). Flo isil® was supplied by Sigma-Ald ich (S . Louis, MO, USA). Comme cial LAS consis o a mix u e o ou alkyl chain homologues, C10 o C13, wi h an a e age alkyl chain leng h o app oxima ely C11.6 [5]. The comme cial sample, which con ained LAS-C10 (10.3 %), LAS- C11 (35.0 %), LAS-C12 (35.1 %) and LAS-C13 (19.6 %) was supplied by CEPSA Química (Mad id, Spain). Wo king solu ions we e p epa ed by dilu ion o a s ock s anda d solu ion (4000 mg/L, LAS mix u e) in MeOH:H 2 O (1:1, / ). 2.2. Ins umen a ion and so wa e Ch oma og aphic analyses we e pe o med on an Agilen 1200 se ies HPLC (Agilen , USA) equipped wi h a acuum degasse , a bina y pump, an au osample , and a he mos a ed column compa men . Th ee ypes o ch oma og aphic columns we e es ed, a Zo bax Eclipse XDB-C18 (50 mm x 4.6 mm i.d., 1.8 µm pa icle size) acqui ed om Agilen (Ba ce- lona, Spain), an Ine sil Ph-3 (150 mm x 4 mm i.d., 5.0 µm pa icle size) om GL Science (Dillenbu gs aa , The Ne he lands), and, a Kine ex Pola C18 (50 mm x 3.0 mm i.d., 2.6 µm pa icle size) supplied by Phenomenex (Cali o nia, USA). The HPLC sys em was coupled o a 6410 iple quad upole mass spec ome e equipped wi h an elec osp ay ioniza ion sou ce (ESI) (Agilen , USA). Ioniza ion o analy es was ca ied ou using he ollowing se ings: MS capilla y ol age 4000 V, d ying-gas low a e 9 L/min, d ying-gas empe a u e 350 ◦C, and nebulize p essu e 40 psi. A o ex-mixe (Velp Scien i ica, Usma e, I aly), an ul asound Sono ex digi ec ba h (Selec a, Be lín, Ge many), a Mac o onic BLT cen i uge (Selec a, Ab e a, Spain), a Dionex ASE 350 accele a ed Sol- en Ex ac o (Dionex, Sunny ale, USA) and a sample concen a o (S ua , S a o dshi e, UK) we e used o sample ea men . S a g aphics Cen u ion Ve sion 18 (S a poin Technologies Inc., Wa en on, VA, Fig. 4. Ex e nal calib a ion, s anda d addi ion and ma ix-ma ched calib a ion cu es ob ained o compos sample. J. Ma ín e al. Mic ochemical Jou nal 204 (2024) 111089 4 USA) was used o s a is ical ea men o da a. 2.3. Sample collec ion and p e ea men P ima y, seconda y, and anae obically diges ed and dehyd a ed sludge samples we e collec ed om one u ban WWTPs in Paloma es del Río (Se ille, Spain). Compos samples we e collec ed in a 1-yea sam- pling pe iod collec ed om a compos ing plan whe e anae obically diges ed sludge was ea ed in he mally con olled dynamic ba e ies wi h ae a ion p o ided by u ning. Soil sample was Medi e anean ype and was collec ed om an ag icul u al land in Se ille. Samples we e eeze-d ied in a C yodos-50 lyophilize (Tels a , Spain), homogenized in a mo a , and sie ed (pa icle size <1 mm). 2.4. Sample ea men Final op imized sample ea men was as ollows: lyophilized sam- ples (0.25 g dw) we e ans e ed o glass cen i uge ubes wi h 0.5 g o lo isil and we e homogenized. Tubes we e b ie ly o ex-mixed using 8 mL o MeOH and sonica ed in an ul asonic ba h o 5 min. A e ex ac ion, ubes we e cen i uged o 10 min a 2900 ×g. The liquid phase was ans e ed o a clean ube and he ex ac ion was epea ed. The combined liquid phase o wo ex ac ions was dilu ed wi h MeOH: H 2 O (1:1, : ) o a inal olume o 25 mL o compos and soil, o 100 mL o seconda y sludge and o 100 mL o p ima y and diges ed sludge o which 0.5 mL we e ans e ed o 10 mL using a 1:1, : MeOH:H 2 O solu ion. The liquid phase was il e ed h ough a 0.22 µm cellulose sy inge il e and collec ed in a ial o LC–MS/MS de e mina ion. 2.5. LC-MS/MS de e mina ion Ch oma og aphic sepa a ion was ca ied ou in an Ine sil Ph-3 (150 mm x 4 mm i.d., 5.0 µm pa icle size) om GL Science (Dillenbu gs aa , The Ne he lands) p o ec ed by an Ine sil Ph-3 Ca idge Gua d Column E (10 mm x 4 mm i.d., 5.0 µm pa icle size) (GL Science, The Ne he lands). Mobile phase was composed o 10 mM ammonium ace- a e (sol en B) and MeOH (sol en A). The g adien p og am was as ollows: 0–5 min, linea g adien om 70 o 80 % o sol en A and held o 2 min, back o ini ial condi ions in 1 min and held o 2 min o eequilib a ion. Flow a e was ixed a 0.6 mL/min. The injec ion ol- ume was 10 µL and he column empe a u e was main ained a 35 ◦C. LAS homologues we e analyzed in mul iple eac ion moni o ing (MRM) mode. Two MRM ansi ions we e selec ed o each LAS homologue (C10-C13) o quan i ica ion and con i ma ion. LAS homologues we e all ionized in nega i e mode. The quan i a ion/con i ma ion ions and he op imized agmen o and collision ene gy o each analy e a e shown in Table 1. 3. Resul s and discussion 3.1. Me hod op imiza ion The op imiza ion o he me hodology applied o he analysis o LAS in sludge samples was ca ied ou o e wo s eps o he sample p o ocol: Table 2 Me hod de ec ion limi s (MDLs) and me hod quan i ica ion limi s (MQLs) o LAS C10-C13 in soil, compos and sludge samples. MDL (mg/kg dw) MQL (mg/kg dw) C10 C11 C12 C13 C10 C11 C12 C13 Soil 0.006 0.018 0.021 0.012 0.02 0.06 0.07 0.04 Compos 0.006 0.021 0.021 0.012 0.02 0.07 0.07 0.04 Diges ed sludge 0.246 0.846 0.861 0.453 0.82 2.82 2.87 1.51 Seconda y sludge 0.015 0.042 0.036 0.018 0.05 0.14 0.12 0.06 P ima y sludge 0.258 0.867 0.858 0.465 0.86 2.89 2.86 1.55 Table 3 Reco e y assay, p ecision and ueness o LAS C10-C13 in soil, compos and sludge ma ices. Soil Low le el Medium le el High le el Spiked Found RSD Rec Spiked Found RSD Rec Spiked Found RSD Rec (mg/kg dw) (mg/kg dw) (%) (%) (mg/kg dw) (mg/kg dw) (%) (%) (mg/kg dw) (mg/kg dw) (%) (%) LAS C10 0.52 0.48 10.9 93.2 14.4 13.4 3.10 92.8 82.4 85.1 2.57 103 LAS C11 1.75 1.95 25.3 111 49.0 46.9 3.99 95.7 280 302 2.72 108 LAS C12 1.76 1.85 23.8 105 49.1 43.9 3.86 89.3 281 288 3.09 103 LAS C13 0.98 0.96 21.2 97.6 27.4 24.7 6.29 90.0 157 158 2.57 101 Compos LAS C10 4.12 4.57 2.70 111 20.6 19.7 4.42 95.5 82.4 82.4 5.55 100 LAS C11 14.0 13.9 19.7 99.5 70.0 65.8 3.67 94.0 280 284 5.77 101 LAS C12 14.0 13.9 13.0 98.7 70.2 64.6 4.18 92.0 281 290 2.87 103 LAS C13 7.84 7.57 11.6 96.5 39.2 38.2 3.91 97.5 157 167 3.83 106 Diges ed sludge LAS C10 824 824 1.39 100 8240 7762 5.05 94.2 16,480 16,974 3.55 103 LAS C11 2800 2778 2.65 99.2 28,000 27,076 1.98 96.7 56,000 57,120 2.95 102 LAS C12 2808 2745 0.54 97.8 28,080 26,957 3.28 96.0 56,160 57,283 2.86 102 LAS C13 1568 1631 5.18 104 15,680 17,875 2.07 114 31,360 36,064 2.81 115 Seconda y sludge LAS C10 8.24 6.98 4.94 84.8 41.2 40.9 4.95 99.3 165 161 6.72 97.4 LAS C11 28.0 27.7 0.15 99.0 140 133 1.09 95.0 560 542 3.52 96.7 LAS C12 28.1 32.5 1.02 116 140 137 1.00 97.8 562 532 3.14 94.7 LAS C13 15.7 21.8 0.45 139 78.4 80.8 2.45 103 314 287 3.18 91.6 P ima y sludge LAS C10 824 790 1.39 95.8 8240 7482 5.05 90.8 16,480 16,480 3.55 100 LAS C11 2800 2713 2.65 96.9 28,000 25,816 1.98 92.2 56,000 56,000 2.95 100 LAS C12 2808 2757 0.54 98.2 28,080 25,665 3.28 91.4 56,160 56,160 2.86 100 LAS C13 1568 1582 5.18 101 15,680 14,394 2.07 91.8 31,360 31,360 2.81 100 J. Ma ín e al. Mic ochemical Jou nal 204 (2024) 111089 5 1) he ch oma og aphic sepa a ion and LAS de e mina ion and 2) he sample ea men (ex ac ion and pu i ica ion o analy es om he sludge ma ix). 3.1.1. Op imiza ion o he ch oma og aphic de e mina ion Th ee p ima y a iables we e conside ed o achie e a good esolu- ion o he peaks, adequa e selec i i y, and sensi i i y: he analy ical column, he composi ion o he mobile phase and he de ec ion condi ions. The op imiza ion o he MS/MS condi ions was pe o med by di ec injec ions (wi hou column) o a mix u e s anda d solu ion a 1.5 mg/L in MeOH:H 2 O a a low a e o 0.6 mL/min by using MassHun e op i- mize so wa e. A e ob aining m/z da a o p ecu so ion and p oduc ions, di e en mobile phases we e es ed in he a emp o ob ain he bes ioniza ion o compounds and o achie e highe esponses. MeOH and ACN we e e alua ed as o ganic mobile phases. As aqueous sol en , deionized wa e wi h di e en addi i es ( o mic acid and ammonium ace a e) was checked. Since LAS showed highe esponses in nega i e ioniza ion mode a g adien elu ion wi h MeOH (sol en A) and a bu e solu ion ammonium ace a e (10 mM) (sol en B) was selec ed as mobile phase. When MS/MS condi ions and mobile phase we e selec ed, di e en analy ical columns (Zo bax Eclipse XDB-C18 (50 mm x 4.6 mm i.d., 1.8 µm pa icle size), Ine sil Ph-3 (150 mm x 4 mm i.d., 5.0 µm pa icle size) and Kine ex Pola C18 (50 mm x 3.0 mm i.d., 2.6 µm pa icle size) we e used o ob ain good esolu ion o he peaks. The op imal ch oma og aphic condi ions we e selec ed acco ding o he peak esolu ion and asymme y ac o s. F om Fig. S1 we can see ha he Ine sil Ph-3 column p o ided a be e peak shape in a sho ime. Al hough wide-bo e columns and, consequen ly, highe low a es esul in lowe ESI e iciency and educed MS sensi i i y, he Ine sil-Ph3 wide- bo e column (150 mm x 4 mm i.d., 5.0 µm pa icle size) p o ided be e peak symme y in a compa able un ime han he Kine ex Pola C18 (50 mm x 3.0 mm i.d., 2.6 µm pa icle size) and he Zo bax Eclipse XDB-C18 (50 mm x 4.6 mm i.d., 1.8 µm pa icle size). Du ing he op imiza ion s ep, he middle-bo e column p esen ed sligh ly ailing peaks o LAS homologues. Fu he mo e, he Ine sil-Ph3 gene a ed a highe signal o LAS homologues han he o he wo columns, hus making he wide-bo e column he p e e ed choice. In addi ion, his column gene a ed lowe p essu es (up o 200 ba ) han o he columns. 3.1.2. Op imiza ion o he ex ac ion and pu i ica ion condi ions Fi s o all, pu i ica ion condi ions we e op imized. Th ee so ben s o di e en na u e, i.e. one e e sed-phase so ben (C18), one mixed mode so ben (PSA) and a no mal-phase so ben (Flo isil), we e e alua ed as clean-up so ben s o dec ease ma ix e ec s. The expe imen was ca ied ou in iplica e using aliquo s o 1 g o compos spiked wi h a LAS mix u e a a concen a ion le el o 15 mg/kg dw. Fo op imiza ion, a gene al and non-op imized sample ex ac ion me hod was used a i s : Aliquo s o samples (1 g) we e weighed in o 12 mL glass ials. The samples we e o exed o homogeniza ion in 8 mL o MeOH and cen i uged o 10 min a 4050 ×g. The sol en ex ac was ans e ed o a clean ube and subjec ed o a clean-up s ep by dSPE adding 1 g o each so ben in each case. The ubes we e shaken igo ously o 1 min and cen i uged a 4050 ×g o 15 min. The sol en was il e ed h ough a 0.22 µm nylon and dilu ed up o 10 mL using wa e . A 10 µL aliquo o he ex ac was injec ed in o he LC ins umen . Fig. 1 summa izes he esul s ob ained. LAS C10-C13 exhibi ed a simila beha iou , poo eco e ies wi h PSA p obably explained by hei e en ion in he so ben h ough elec os a ic in e ac ions, whe eas a signi ican dec ease o he signal was obse ed wi h C18, which can be a ibu ed o i s highe e en ion by pa i ioning as consequence o i s simila in e ac ions. Accep able eco e ies we e ob ained using lo isil, inally chosen as he clean-up so ben ma e ial. Flo isil can emo e he majo pola in e e ences elu ing a he same ime as he LAS homo- logues. The e o e, lo isil was selec ed as clean-up so ben o he nex op imiza ion expe imen s. Di e en ex ac ion echniques (ul asound-assis ed ex ac ion (UAE ollowed by a clean-up by dSPE (UAE +dSPE)); UAE and dSPE com- bined in a single s ep (UAE-dSPE); and selec i e p essu ized liquid ex ac ion (SPLE)) we e assessed using lo isil as clean-up so ben in all cases (Fig. 2). The simul aneous ex ac ion and clean-up s a egy used in UAE-dSPE and SPLE is in conco dance wi h he ou h p inciple o G een Table 4 Summa y o analy ical me hods o he de e mina ion o LAS homologues in sludge. Ex ac ion me hod Sludge amoun (g) Ex ac ion sol en Sol en olume (mL) Ex ac ion ime Reco e y (%) MQL (mg/kg dw) Calib a ion De e mina ion Re e ence Soxhle No da a MeOH No da a 8 h 85–99 No da a EC LC-UV [22] P essu ized-liquid ex ac ion 10 MeOH:Wa e (9:1, / ) 22 3 ×5 min 52–85 0.025 EC LC-MS/MS [23] Mic owa e- assis ed ex ac ion 0.5 MeOH 5 10 min 83–102 0.85–4.40 (DAD) 1.11–6.09 (FLD) EC LC-UV-DAD/ FLD [24] Ul asound- assis ed ex ac ion No da a MeOH 20 3 ×20 min 78–93 0.0002–100 (MDL) EC LC-MS [25] Ul asound- assis ed ex ac ion 1 MeOH 50 25 min 48–99 13–56 (UV- DAD) 5–18 (FLD) EC LC-UV-DAD/ FLD [26] P obe sonica ion ex ac ion 0.5 MeOH 10 7 min 84–97 2.78–15.22 EC LC-UV-DAD/ FLD [27] Mic owa e- assis ed ex ac ion 5 MeOH 25 25 min No da a 0.011–0.018 EC LC-FLD [28] QuEChERS me hod 0.5 ACN 10 No da a 89–114 0.002–1 MM LC-MS/MS [20] Ul asound- assis ed ex ac ion 0.25 MeOH 16 2 x 5 min 85–115 0.02–2.89 SA LC-MS/MS P oposed me hodology ACN: ace oni ile; EC: ex e nal calib a ion; DAD: diode a ay de ec o ; DAD-UV: diode a ay de ec o -ul a iole ; FLD: luo escence de ec o ; LC-FLD: liquid ch oma og aphy- luo escence de ec ion; LC-MS: liquid ch oma og aphy-mass spec ome y; LC-MS/MS: liquid ch oma og aphy- andem mass spec ome y; LC-UV- DAD/FLD: liquid ch oma og aphy-ul a iole -diode a ay de ec o - luo escence de ec ion; MDL: me hod de ec ion limi s; MeOH: me hanol; MQL: me hod quan i i- ca ion limi s; MM: ma ix-ma ched calib a ion; SA: s anda d addi ion. J. Ma ín e al. Mic ochemical Jou nal 204 (2024) 111089 6 Analy ical Chemis y, in eg a ion o analy ical p ocesses and ope a ions sa es ene gy and educes he use o eagen s [18]. To simpli y he ex ac ion p ocedu e, and acco ding o he esul s o Fig. 2 he combina ion o UAE-dSPE was selec ed in a single s ep. Sligh ly highe ex ac ion eco e ies we e achie ed o he LAS mix u e in addi ion o he signi ican educ ion in ime o he clean s ep in he sample p o ocol (mean eco e ies o 81, 76 and 75 % o UAE-dSPE, UAE +d-SPE, and SPLE, espec i ely). The ex ac ion ime was se o 5 min in all cases o ob ain homogeniza ion o he ex ac ed samples. Once UAE-dSPE echnique and lo isil so ben we e ixed as ex ac- ion echnique and clean-up so ben and, o inc ease ex ac ion e i- ciency and g eenness o he me hod, he sample amoun , lo isil amoun and ex ac ion cycles we e op imized by using a Box–Behnken expe i- men al design. The design ma ix o he Box-Behnken s udy was gene a ed using h ee le els o each o he h ee ac o s: sample amoun (0.25, 0.625 and 1 g), ex ac ion s eps (1, 2 and 3) and lo isil amoun (500, 1250 and 2000 mg). The design ma ix is shown in Table S1. A o al o 15 analy ical assays we e ca ied ou . All expe imen s we e pe o med in a andomized o de o minimize he e ec s o uncon olled a iables ha may in oduce a bias in he measu emen s. Th ee cen al poin expe imen s we e inco po a ed o es ima e he expe imen al e o ha does no depend on he i ed model. To be e unde s and he in e ac ions o independen a iables on he ex ac ion esul s, he h ee-dimensional esponse su ace images and co esponding con ou plo s we e isualized. Responses o each com- pound in he expe imen s o he Box–Behnken design we e i s no malized be ween 0 and 1, and he global desi abili y unc ion was de ined as hei geome ic mean. The plo o his unc ion: cycles s so ben amoun (A), sample amoun s so ben amoun (B), and cycles s sample amoun (C) a e shown in Fig. 3. The cycles and he sample amoun esul ed in he mos signi ican a iables, being no ewo hy he posi i e in e ac ion in he o me and he nega i e in e ac ion o he sample amoun . When a la ge quan i y o sludge was used, a highe dec ease in esponse was obse ed due o he conside able ma ix e ec . Rega ding he cycles, wo s eps we e signi ican o inc ease he ex ac ion eco e ies. Acco ding o he esul s, 0.25 g o sample, 0.5 g o lo isil and wo ex ac ion s eps we e selec ed. The esul s o he inal op imized me hod can be seen abo e in sec ion 2.4. 3.2. Analy ical pe o mance 3.2.1. Ma ix e ec assessmen Al hough he e alua ion o he in luence o di e en ma ices o an analy ical me hod is a manda o y s ep in he me hod’s alida ion, i s applicabili y o he moni o ing o ace compounds in complex sludge samples is no simple, no is i pa o he ou ine o mos labo a o ies. This sec ion ocusses on he accu a e p ocedu e o quan i ica ion con- cen a ions o LAS in di e en ypes o ma ices, namely p ima y sludge, seconda y sludge, diges ed sludge, compos , and soil. To assess he in luence o he ma ix in he quan i ica ion p ac ice, h ee calib a ion cu es we e p epa ed o each compound: one in pu e sol en (ex e nal calib a ion, EC), he second one using ma ix-ma ched wi h comme cial e ilize samples (ma ix-ma ched calib a ion, MM), and he las one using s anda d addi ion (SA) me hod o each ype o ma ix (soil, compos , and sludges). Calib a ion cu es we e cons uc ed using he LAS peak a ea a io e sus he LAS homologue concen a ion. Calib a ion s anda ds we e p epa ed a eigh di e en analy e con- cen a ion le els. Taking in o accoun he LAS speci ic esponses and he concen a ion le els p esen in sludge samples, di e en concen a ion anges we e used o each ma ix ( om 0.1 o 100 mg/kg dw o soil; om 5 o 800 mg/kg dw o compos and comme cial e ilize ; om 10 o 1600 mg/kg dw o seconda y sludge and om 200 o 240000 mg/kg dw o p ima y and diges ed sludge o he comme cial LAS sample). No e ha a di e en dilu ion ac o was also applied depending on he ype o ma ix (1:200 o soil, comme cial e ilize , and compos , 1:400 o seconda y sludge and 1:8000 o diges ed and p ima y sludge). Fo MM and SA calib a ions, spiked samples we e o exed o 2 min and hen le o s and o 24 h a 4 ◦C in he da k be o e analysis. This allows he analy es o come in o ull con ac wi h he sample. Ideally, a ma ix-ma ched s anda d should no con ain any o he selec ed com- pounds, bu in p ac ice his is no always possible. The a eas o com- pounds ha a e p esen in he blank ex ac mus be sub ac ed o he a ea ob ained om he ma ix-ma ched s anda ds. Table S2 shows he linea i y assessmen ob ained in he wo king ange o LAS in di e en ypes o calib a ions and sludge samples. Fig. 4 shows calib a ions cu es ob ained o compos sample wi h EC, MM and SA. A S uden ’s - es was applied o compa e he slope o he calib a ion cu es: EC s. MM; EC s. SA; and MM s. SA. The absence o ma ix e ec co esponds o slope EC =slope MM; slope EC =slope SA; o slope MM =slope SA. This mus be checked o s a is ical signi icance. The calcula ed is compa ed wi h he wo- ailed abula ed alue, ab o he app op ia e numbe o deg ees o eedom a P% con idence. Typical alues a e k =2 o 95 % con idence, so, i <k, he a io o he slopes is no signi ican ly di e en om 1, and, i >k, he a io o he slopes is signi ican ly di e en om 1 and he s anda d addi ion calib a ion mus be used. The calcula ed S uden ’s (2.027–22.435) (Table S3) showed s a is ical di e ences among he slope alues o he calib a ion cu es and, consequen ly, he use o s anda d addi ion calib a ion was equi ed o minimize ma ix e ec s as hese in e e e wi h analy e measu emen signals. As can be seen, calcula ed S uden ’s inc eases wi h he complexi y o he ma ix ob aining highe alues o p ima y (8.804–12.994), seconda y (5.714–21.909) and diges ed sludge (10.406–22.435). Table 5 Quan i ica ion o LAS (mg/kg dw) in soil, comme cial e ilize , compos and di e en sludge samples by ex e nal, ma ix-ma ched and s anda d addi ion calib a ions. Rela i e s anda d de ia ion pe cen age (% RSD) is shown in b acke s. Soil C10 C11 C12 C13 Ex e nal calib a ion 0.52 (5.8) 3.50 (8.3) 3.64 (2.2) 1.94 (2.5) Ma ix-ma ched calib a ion 0.74 (5.5) 5.07 (3.0) 6.62 (1.4) 3.82 (1.6) S anda d addi ion 0.06 (14) 0.29 (14) 0.36 (8.8) 1.59 (8.3) Comme cial e ilize C10 C11 C12 C13 Ex e nal calib a ion 0.93 (9.9) 5.44 (1.9) 5.56 (3.0) 3.25 (0.8) Ma ix-ma ched calib a ion 1.27 (9.4) 7.38 (1.7) 8.70 (2.1) 5.23 (0.6) S anda d addi ion 1.07 (2.1) 4.93 (4.8) 5.05 (7.8) 5.97 (1.8) Compos C10 C11 C12 C13 Ex e nal calib a ion 3.54 (9.4) 19.6 (13) 44.1 (18) 49.6 (17) Ma ix-ma ched calib a ion 4.59 (9.2) 24.3 (12) 50.3 (17) 55.3 (16) S anda d addi ion 4.29 (11) 33.7 (15) 76.8 (19) 95.9 (18) Diges ed sludge C10 C11 C12 C13 Ex e nal calib a ion 255 (7.6) 1438 (7.1) 2197 (6.2) 1537 (7.1) Ma ix-ma ched calib a ion 328 (7.5) 1754 (7.0) 2478 (5.9) 1731 (6.8) S anda d addi ion 696 (6.9) 2365 (5.7) 4395 (4.1) 3401 (4.9) Seconda y sludge C10 C11 C12 C13 Ex e nal calib a ion 17.0 (18) 68.4 (8.8) 120 (8.4) 164 (8.7) Ma ix-ma ched calib a ion 21.9 (17) 83.6 (8.6) 135 (8.0) 181 (8.6) S anda d addi ion 33.3 (4.4) 135 (4.4) 191 (2.0) 240 (3.2) P ima y sludge C10 C11 C12 C13 Ex e nal calib a ion 223 (26) 1223 (26) 1606 (34) 1033 (33) Ma ix-ma ched calib a ion 288 (26) 1497 (26) 1841 (32) 1186 (31) S anda d addi ion 430 (26) 2674 (22) 4120 (30) 3029 (30) J. Ma ín e al. Mic ochemical Jou nal 204 (2024) 111089 7 The ex e nal s anda d calib a ion has been he mos used app oach o de e mine analy e concen a ions in analy ical p ocedu es [19]. Howe e , i s accu acy hinges on he absence o signi ican in e e ences in he sample, a condi ion ha is o en challenging o mee , pa icula ly in complex samples like sludge. Ma ix-ma ched calib a ion cu es necessi a e a simila ma ix ee om he a ge compounds, e ec i ely se ing as a blank sample. Un o una ely, o subs ances like LAS, inding a sui able blank ma ix is nea ly impossible due o i s ubiqui ous p esence in sewage sludge. Mo eo e , since LAS homologues a e gene ally p esen in soil, due o his o ic use o sludge-amendmen , and compos samples, due o he equen addi ion o sludge in compos ing, he s anda d addi ion me hod is desi able. Va ious analy ical ap- p oaches ha e been epo ed o assessing LAS concen a ions in sludge samples, wi h he p e alen use o EC. In con as , Be g´ e e al. (2016) [20] in oduced a dis inc i e me hodology ha employs syn he ic ma ices (MM) o LAS quan i ica ion. Ou esul s e eal ha he ma ix e ec has he po en ial o al e concen a ion ou comes when employing EC. Simul aneously, i is wo h no ing ha MM, while employed, may also in oduce e o s owing o he inhe en dissimila i y be ween he ac ual ma ices and he syn he ic coun e pa s, which a e no a pe ec ma ch. 3.2.2. Me hod alida ion The op imized me hod was alida ed acco ding o Eu achem Guide [21]. Ins umen al limi s o quan i ica ion (LOQ) we e lowe han 1 ng/ mL o he comme cial LAS sample (calcula ed om he signal- o-noise a io =10 injec ing sol en o i ied wi h dec easing amoun s o LAS). Limi s o de ec ion (LODs) we e calcula ed om he signal- o-noise a io =3 injec ing sol en s o i ied wi h dec easing amoun s o LAS homo- logues. The me hod quan i ica ion limi s (MQLs), and me hod de ec ion limi s (MDLs) we e calcula ed based on he LOQs and LODs, espec- i ely, sample weigh , dilu ion ac o o each ype o sample, and mean eco e ies o LAS a low concen a ion le el (Table 2). The limi o quan i ica ion enables he measu emen o LAS concen a ions up o 1000 imes lowe han he concen a ion limi s s ipula ed in he EU di ec i e d a . The concen a ion ange o calib a ion cu es applied o he sludge ex ac s ul illed all he s a is ical equi emen s o a linea me hod (Table S2). Fo all compounds, a well-de ined wo king ange was ob- ained, being cha ac e ized by R 2 ≥0.996 using sol en , R 2 ≥0.994 ma ix-ma ched wi h comme cial e ilize and soil s anda d addi ion me hod, R 2 ≥0.922 wi h compos s anda d addi ion me hod, R 2 ≥0.956 wi h seconda y sludge s anda d addi ion me hod, R 2 ≥0.991 wi h diges ed sludge s anda d addi ion me hod and R 2 ≥0.985 wi h p ima y sludge s anda d addi ion me hod. Linea ange o LAS homologues a ied om 0.1 o 100 mg/kg dw o soil; om 5 o 800 mg/kg dw o compos ; om 10 o 1600 mg/kg dw o seconda y sludge and om 200 o 240000 mg/kg dw o p ima y and diges ed sludge. The accu acy o he p oposed me hod was alida ed in e ms o ueness and p ecision. Fo i ied sludge, soil, and compos samples a h ee concen a ions le els (low, medium, and high) we e analyzed (Table 3). P ecision, exp essed as ela i e s anda d de ia ion (%, RSD) was de e mined om iplica e spiked samples du ing di e en days, and he ueness was e alua ed using he eco e y da a by compa ing he ound s. spiked le el da a. The accu acy o he me hod was sa is- ac o y o all he analy es wi h ela i e eco e ies alues anging om 84.8 % o 139 % and wi h an RSD <26 % and <7 % a low and high le el, espec i ely. Da a indica e ha he me hodology o de e mine LAS in sludge samples using SA calib a ion is accu a e, and ha he p esence o co-ex ac ed ma ix componen s, which could supp ess/enhance he analy e signal in mass spec ome y, did no a ec he pe o mance o he me hod. The speci ici y o he me hod was de e mined by compa ing he ch oma og ams o blank wi h he co esponding spiked sample. No in- e e ences om endogenous subs ances we e obse ed a he e en ion ime o he analy es elu ed. These inds sugges ha he spec ome ic condi ions ensu ed high selec i i y o he LC–MS/MS me hod (Fig. S2 and S3). Table 4 p esen s a concise o e iew o analy ical app oaches p e i- ously documen ed o assessing LAS in sludge. Repo ed me hods did no pe o med any clean-up s ep while in he p oposed me hodology he ex ac ion and clean-up we e done simul aneously, which is manda o y o dec ease ma ix e ec . No ably, he Soxhle me hod [22] demanded he leng hies p ocessing ime (8 h), while p essu ized-liquid ex ac ion [23] equi ed he la ges sample quan i y (10 g). MeOH eme ged as he sol en o choice o LAS ex ac ion, al hough he QuEChERS me hod [20] necessi a ed he use o ACN. The p oposed me hodology u ilized he smalles sample size among epo ed me hods and employed ex ac ion imes compa able o o sho e han hose ound in he li e a u e. This is pa icula ly no able gi en ha he majo i y o he me hods ci ed in he li e a u e did no inco po a e a clean-up s ep. Mic owa e-assis ed ex ac ion [24] me hodology employed he lowes sol en olume. Mos o he me hodologies de eloped used ex e nal calib a ion o quan i ica ion. Ob ained eco e ies we e simila o hose ob ained in li e a u e. Rega ding MQLs, alues ob ained in his wo k (0.02–2.89 mg/kg dw) we e simila o hose me hods ha employs MS de ec o (0.025 mg/kg dw [23]) and lowe han hose ob ained wi h UV/FLD: 0.85–4.40 mg/kg dw [24], 5–56 mg/kg dw [26] o 2.78–15.22 mg/kg dw [27]. 3.3. Me hod applica ion Me hod was applied o soil, comme cial e ilize , compos , p ima y, seconda y, and diges ed sludge using EC, MM and SA as calib a ion me hods (n =5). Resul s a e shown in Table 5. No ably, p ima y and diges ed sludge exhibi ed he highes concen a ions, whe eas soil dis- played he lowes concen a ion, a ibu able o a dilu ion e ec . Table 5 e eals ha C13 concen a ions in soil a e qui e simila when employing EC and SA calib a ion me hods (1.94 and 1.59 mg/kg dw, espec i ely). Howe e , in he case o p ima y sludge, C13 concen a- ions using SA a e h ee imes highe han hose ob ained using EC (1033 and 3029 mg/kg dw o EC and SA, espec i ely). These indings un- de sco e he conside able impac o he chosen calib a ion me hod on he quan i ica ion o LAS in soil, compos , and sludge samples, especially in mo e complex samples like p ima y, seconda y, and diges ed sludge. This highligh s he impo ance o selec ing an app op ia e calib a ion me hod o ensu e accu a e quan i ica ion. I is wo h no ing ha he o al concen a ion o LAS in ea ed sludge such as compos (210.69 mg/kg dw using SA calib a ion) does no exceed he limi s ou lined in he EU di ec i e d a o land applica ion o sludge (2,600 mg/kg). 4. Conclusions A me hod o he de e mina ion o LAS homologues C10-C13 in p ima y, seconda y, diges ed sludge, compos , and soil was op imized and alida ed. Sample ea men is based on easy- o-pe o m, cos - e ec i e and use - iendly p ocedu e ha combine he ex ac ion and clean-up in a single s ep (UAE-dSPE). The me hod simpli ies bo h ex ac ion and clean-up, condensing hem in o a single s ep while educing ma ix e ec s h ough a dilu ion s a egy, aligning wi h he p inciples o G een Analy ical Chemis y. Analy ical de e mina ion is ca ied ou by LC–MS/MS. The in luence o di e en ma ices esul ed a signi ican ea u e in he me hod’s alida ion. The me hod o s anda d addi ion is he quan i a i e analysis p ocedu e selec ed o minimize ma ix e ec s ha can in e e e wi h analy e measu emen signals. I is key o quali y con ol, in e ms o moni o ing pu poses since ma ix e ec may in oduce a bias in he calcula ed concen a ion. The me hod accu acy in e ms o ela i e eco e ies was highe han 84.5 % in all kinds o sludge samples. The limi s o quan i ica ion allow he mea- su emen o LAS concen a ions 1000 imes lowe han he concen a- ion limi s ixed by he EU di ec i e d a o land applica ion o sludge J. Ma ín e al. Mic ochemical Jou nal 204 (2024) 111089 8 (2,600 mg/kg). The p oposed me hod cons i u es a use ul and accu a e ool o e alua e LAS dis ibu ion along sludge s abiliza ion ea men s. CRediT au ho ship con ibu ion s a emen Julia Ma ín: W i ing – e iew & edi ing, Me hodology, Concep u- aliza ion. Ca men Mejías: Visualiza ion, In es iga ion. Juan Luis San os: Valida ion, Me hodology, Fo mal analysis. I ene Apa icio: W i ing – e iew & edi ing, Valida ion, Resou ces. Es eban Alonso: Supe ision, P ojec adminis a ion, Funding acquisi ion, Concep uali- za ion. John Heinze: P ojec adminis a ion, Visualiza ion. Decla a ion o compe ing in e es The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape . Da a a ailabili y Da a will be made a ailable on eques . Acknowledgmen s Au ho s acknowledge he inancial suppo om he Council o LAB/LAS En i onmen al Resea ch (CLER). C. Mejías g a e ully hanks Uni e si y o Se ille o he p edoc o al con ac (g an numbe : VI PPIT-US 2021 II.2A). Appendix A. Supplemen a y da a Supplemen a y da a o his a icle can be ound online a h ps://doi. o g/10.1016/j.mic oc.2024.111089. Re e ences [1] Eu opean Union. Wo king documen on sludge, hi d d a , 27 Ap il 2000. Eu opean Union, B ussels. h ps://www.isp ambien e.go .i /i /p oge i/ca ella- p oge i-in-co so/suolo-e- e i o io-1/uso-dei- anghi-di-depu azione-in- ag icol u a-a i i a-di-con ollo-e- igilanza-del- e i o io/ iles/3 d_D a _sludge_ en.pd . [2] C. Mejías, J. Ma ín, J.L. San os, I. Apa icio, E. Alonso, Occu ence o Pha maceu icals and Thei Me aboli es in Sewage Sludge and Soil: A Re iew on Thei Dis ibu ion and En i onmen al Risk Assessmen , T ends En i on. Anal. Chem. 30 (2021) e00125, h ps://doi.o g/10.1016/j. eac.2021.e00125. [3] M.M. Gonz´ alez, J. Ma ín, D. Camacho-Mu˜ noz, J.L. San os, I. Apa icio, E. Alonso, Deg ada ion and en i onmen al isk o su ac an s a e he applica ion o compos sludge o he soil, Was e Manag. 32 (7) (2012) 1324–1331, h ps://doi.o g/ 10.1016/j.wasman.2012.02.023. [4] Di ec i e 86/278/EEC on he p o ec ion o he en i onmen , and in pa icula o he soil, when sewage sludge is used in ag icul u e: Using sewage sludge in a ming. [5] HERA P ojec , Human and En i onmen al Risk Assessmen on ing edien s o Household Cleaning P oduc s. LAS, Linea Alkylbenezene Sulphona e (CAS No. 68411-30-3). Re ised HERA Repo , Ap il 2013. h ps://www.he ap ojec .com/ iles/HERA-LAS e ised Ap il 2013 Final1.pd . [6] G. Mininni, A.R. Blanch, F. Lucena, S. Be selli, EU policy on sewage sludge u iliza ion and pe spec i es on new app oaches o sludge managemen , En i on. Sci. Pollu . Res. 22 (2015) 7361–7374, h ps://doi.o g/10.1007/s11356-014-3132- 0. [7] Y. Yu, S. Wang, P. Yu, D. Wang, B. Hu, P. Zheng, M. Zhang, A bibliome ic analysis o eme ging con aminan s (ECs) (2001–2021): E olu ion o ho spo s and esea ch ends, Sci. To al En i on. 907 (2024) 168116, h ps://doi.o g/10.1016/j. sci o en .2023.168116. [8] Wo ld Heal h O ganiza ion, 1996. P og amme, Uni ed Na ions En i onmen . Linea Alkylbenzene Sul ona es & Rela ed Compounds (En i onmen al Heal h C i e ia Se ies, Volume 169). ISBN 9789241571692. [9] M. Villag asa, M. Guillamon, E. Elja a , D. Ba celo, Ma ix e ec in liquid ch oma og aphy–elec osp ay ioniza ion mass spec ome y analysis o benzoxazinoid de i a i es in plan ma e ial, J. Ch oma og . a. 1157 (1,2) (2007) 108–114, h ps://doi.o g/10.1016/j.ch oma.2007.04.040. [10] A. Cappiello, G. Famiglini, P. Palma, E. Pie ini, V. Te mopoli, H. T u elli, O e coming ma ix e ec s in liquid ch oma og aphy-mass spec ome y, Anal. Chem. 80 (2008) 9343–9348, h ps://doi.o g/10.1021/ac8018312. [11] D. S eine , R. K ska, A. Malacho ´ a, I. Taschl, M. Sulyok, E alua ion o ma ix e ec s and ex ac ion e iciencies o LC-MS/MS me hods as he essen ial pa o p ope alida ion o mul iclass con aminan s in complex eed, J. Ag ic. Food Chem. 68 (2020) 3868–3880, h ps://doi.o g/10.1021/acs.ja c.9b07706. [12] F. Raposo, D. Ba celo, Challenges and s a egies o ma ix e ec s using ch oma og aphy-mass spec ome y: An o e iew om esea ch e sus egula o y iewpoin s, T ends Anal. Chem. 134 (2021) 116068, h ps://doi.o g/10.1016/j. ac.2020.116068. [13] E. Chambe s, D.M. Wag owski-Diehl, Z. Lu, J.R. Mazzeo, Sys ema ic and comp ehensi e s a egy o educing ma ix e ec s in LC/MS/MS analyses. J. Ch oma og ., B Anal. Technol. Biomed, Li e Sci. 852 (2007) 22–34, h ps://doi. o g/10.1016/j.jch omb.2006.12.030. [14] C. Fe e , A. Lozano, A. Agüe a, A.J. Gi ´ on, A.R. Fe n´ andez-Alba, O e coming ma ix e ec s using he dilu ion app oach in mul i esidue me hods o ui s and ege ables, J. Ch oma og . a. 1218 (2011) 7634–7639, h ps://doi.o g/10.1016/j. ch oma.2011.07.033. [15] S.L.R. Ellison, M. Thompson, S anda d addi ions: my h and eali y, Analys 133 (2008) 992–997, h ps://doi.o g/10.1039/b717660k. [16] 2002/657/EC: Commission Decision o 12 Augus 2002 implemen ing Council Di ec i e 96/23/EC conce ning he pe o mance o analy ical me hods and he in e p e a ion o esul s. [17] G.A. Gonz´ alez, M.A. He ado , A p ac ical guide o analy ical me hod alida ion, including measu emen unce ain y and accu acy p o iles, T ends Anal. Chem. 26 (3) (2007) 227–238, h ps://doi.o g/10.1016/j. ac.2007.01.009. [18] C. Mejías, M. A enas, J. Ma ín, J.L. San os, I. Apa icio, E. Alonso, G een Assessmen o Analy ical P ocedu es o he De e mina ion o Pha maceu icals in Sewage Sludge and Soil, C i . Re . Anal. Chem. (2023), h ps://doi.o g/10.1080/ 10408347.2023.2276294. [19] J.T. Sloop, S.C. Allen, J.A. Ca e , G.L. Dona i, B.T. Jones, Ma ix-ma ched wo- poin calib a ion based on he s anda d dilu ion analysis me hod, Mic ochem. J. 160B (2021) 105740, h ps://doi.o g/10.1016/j.mic oc.2020.105740. [20] A. Be g´ e, B. Gi oud, L. Wies , B. Domenjoud, A. Gonzalez-Espinosa, E. Vullie , De elopmen o a mul iple-class analy ical me hod based on he use o syn he ic ma ices o he simul aneous de e mina ion o commonly used comme cial su ac an s in was ewa e by liquid ch oma og aphy- andem mass spec ome y, J. Ch oma og . a. 1450 (2016) 64–75, h ps://doi.o g/10.1016/j. ch oma.2016.04.078. [21] Eu achem. The Fi ness o Pu pose o Analy ical Me hods: A Labo a o y Guide o Me hod Valida ion and Rela ed Topics: Second edi ion (2014). [22] M.T. Ga cía, E. Campos, J. S´ anchez-Leal, I. Ribosa, E ec o linea alkylbenzene sulphona es (LAS) on he anae obic diges ion o sewage sludge, Wa e Res. 40 (2006) 2958, h ps://doi.o g/10.1016/j.wa es.2006.05.033. [23] P. Eichho n, O. L´ opez, D. Ba cel´ o, Applica ion o liquid ch oma og aphy–elec osp ay- andem mass spec ome y o he iden i ica ion and cha ac e isa ion o linea alkylbenzene sul ona es and sul ophenyl ca boxyla es in sludge-amended soils, J. Ch oma og . a. 1067 (2005) 171, h ps://doi.o g/ 10.1016/j.ch oma.2005.01.014. [24] M. Villa , M. Callej´ on, J.C. Jim´ enez, E. Alonso, Op imiza ion and alida ion o a new me hod o analysis o linea alkylbenzene sul ona es in sewage sludge by liquid ch oma og aphy a e mic owa e-assis ed ex ac ion, Anal. Chim. Ac a. 599 (2007) 92, h ps://doi.o g/10.1016/j.aca.2007.07.065. [25] J. Riu, E. Ma ínez, D. Ba cel´ o, A. Gineb eda, L.L. Ti apu, LC–MS de e mina ion o linea alkylbenzene sul ona es and hei ca boxylic deg ada ion p oduc s in in luen and e luen wa e samples and sludges om sewage- ea men plan s, F esenius J. Anal. Chem. 371 (2001) 448, h ps://doi.o g/10.1007/ s002160101052. [26] J.L. San os, I. Apa icio, E. Alonso, A new me hod o he ou ine analysis o LAS and PAH in sewage sludge by simul aneous sonica ion-assis ed ex ac ion p io o liquid ch oma og aphic de e mina ion, Anal. Chim. Ac a 605 (1) (2007) 102–109, h ps://doi.o g/10.1016/j.aca.2007.10.027. [27] M. Villa , R. Fe nandez-To es, M. Callej´ on, P. Villa , J.C. Jim´ enez, Applica ion o p obe sonica ion ex ac ion o he de e mina ion o linea alkylbenzene sul ona es om sewage sludge. Compa ison wi h o he ex ac ion me hods, Mic ochem. J. 90 (2) (2008) 164–170, h ps://doi.o g/10.1016/j.mic oc.2008.05.005. [28] S. Can a e o, A. Za a-Gomez, O. Balles e os, A. Na alon, J.L. Vilchez, C. Ve ge, J. A. De Fe e , Ma ix e ec s udy in he de e mina ion o linea alkylbenzene sul ona es in sewage sludge samples, En i on. Toxicol. Chem. 30 (4) (2011) 813–818, h ps://doi.o g/10.1002/e c.447. J. Ma ín e al.