Salting-out assisted liquid-liquid extracion for biological sample analysis

Grecco CF et al.

Palavras-chave: Salting-out assisted liquid–liquid extraction; biological sample preparation.

ResumoSalting-out assisted liquid-liquid extraction (SALLE) extraction is based on the partition of analyte(s) between water-miscible organic solvent and plasma samples or other biological fluids. The salting-outeffect induces the separation between the phases, organic and aqueous. SALLE is a sample preparation technique that enables the extraction of drugs and their metabolites, including many hydrophilic compounds. This technique allows the obtained extracts to be directly injected into the chromatographic system or by simple dilution. This review presents the theory fundamentals of SALLE technique, the key steps involved in the extraction process and examples of applications of this technique in several areas.

Referências Bibliográficas

[1] Nováková L, Vlčková H. A review of current trends and advances in modern bio-analytical methods: Chromatography and
sample preparation. Anal Chim Acta [Internet]. 2009 Dec;656(1-2):8–35. Available from:
[2] Tang YQ, Weng N. Salting-out assisted liquid–liquid extraction for bioanalysis. Bioanalysis [Internet]. 2013 Jun;5(12):1583–98.
Available from:
[3] Zhang J, Wu H, Kim E, El-Shourbagy TA. Salting-out assisted liquid/liquid extraction with acetonitrile: a new high throughput
sample preparation technique for good laboratory practice bioanalysis using liquid chromatography-mass spectrometry. Biomed
Chromatogr [Internet]. 2009 Apr;23(4):419–25. Available from:
[4] Wu H, Zhang J, Norem K, El-Shourbagy TA. Simultaneous determination of a hydrophobic drug candidate and its metabolite in
human plasma with salting-out assisted liquid/liquid extraction using a mass spectrometry friendly salt. J Pharm Biomed Anal
[Internet]. 2008 Dec;48(4):1243–8. Available from:
[5] Myasein F, Kim E, Zhang J, Wu H, El-Shourbagy TA. Rapid, simultaneous determination of lopinavir and ritonavir in human
plasma by stacking protein precipitations and salting-out assisted liquid/liquid extraction, and ultrafast LC–MS/MS. Anal Chim
Acta [Internet]. 2009 Sep;651(1):112–6. Available from:
[6] Zhang J, Myasein F, Rodila R, Wu H, El-Shourbagy TA. High-throughput salting-out assisted liquid/liquid extraction and
ultrafast LC for same-day delivery of first-in-human bioanalytical data. Bioanalysis [Internet]. 2009 Jul;1(4):715–9. Available
[7] Zhang J, Rodila R, Gage E, Hautman M, Fan L, King LL, et al. High-throughput salting-out assisted liquid/liquid extraction
with acetonitrile for the simultaneous determination of simvastatin and simvastatin acid in human plasma with liquid
chromatography. Anal Chim Acta [Internet]. 2010 Feb;661(2):167–72. Available from:
[8] Yanes EG, Lovett DP. High-throughput bioanalytical method for analysis of synthetic cannabinoid metabolites in urine using
salting-out sample preparation and LC–MS/MS. J Chromatogr B [Internet]. 2012 Nov;909:42–50. Available from: http://
[9]Yang Y, Xu Q, Zhou L, Zhong D, Chen X. High-throughput salting-out-assisted liquid-liquid extraction for the simultaneous
determination of atorvastatin, ortho -hydroxyatorvastatin, and para -hydroxyatorvastatin in human plasma using ultrafast liquid
chromatography with tandem mass spectrometry. J Sep Sci [Internet]. 2015 Mar;38(6):1026–34. Available from: http://doi.wiley.
[10] Xiong X, Yang L. Salting-out-assisted liquid-liquid extraction with acetonitrile for the determination of trimetazidine in rat
plasma using liquid chromatography-mass spectrometry. Biomed Chromatogr [Internet]. 2015 Feb;29(2):268–74. Available
[11] Ahmed S, Mahmoud AM. A novel salting-out assisted extraction coupled with HPLC- fluorescence detection for trace
determination of vitamin K homologues in human plasma. Talanta [Internet]. 2015 Nov;144:480–7. Available from: http://
[12] Sparidans RW, van Hoppe S, Rood JJM, Schinkel AH, Schellens JHM, Beijnen JH. Liquid chromatography–tandem mass
spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid–liquid extraction.
J Chromatogr B [Internet]. 2016 Feb;1012-1013:118–23. Available from:
[13] Qiu Z, Yu J, Dai Y, Yang Y, Lu X, Xu J, et al. A simple LC-MS/MS method facilitated by salting-out assisted liquid-liquid
extraction to simultaneously determine trans -resveratrol and its glucuronide and sulfate conjugates in rat plasma and its
application to pharmacokinetic assay. Biomed Chromatogr [Internet]. 2017 Nov;31(11):e4001. Available from: http://doi.wiley.
[14] Retmana IA, Wang J, Schinkel AH, Schellens JHM, Beijnen JH, Sparidans RW. Liquid chromatography-tandem mass
spectrometric assay for the quantitative determination of the tyrosine kinase inhibitor quizartinib in mouse plasma using saltingout liquid-liquid extraction. J Chromatogr B [Internet]. 2017 Sep;1061-1062:300–5. Available from: http://linkinghub.elsevier.
[15] Horning MG, Boucher EA, Stafford M, Horning EC. A rapid procedure for the isolation of drugs and drug metabolites from plasma.
Clin Chim Acta [Internet]. 1972 Mar;37:381–6. Available from:
[16] Horning MG, Gregory P, Nowlin J, Stafford M, Lertratanangkoon K, Butler C, et al. Isolation of drugs and drug metabolites from
biological fluids by use of salt-solvent pairs. Clin Chem [Internet]. 1974 Feb;20(2):282–7. Available from: http://www.ncbi.nlm.
[17] Bastos ML, Kananen GE, Young RM, Monforte JR, Sunshine I. Detection of basic organic drugs and their metabolites in urine.
Clin Chem [Internet]. 1970 Nov;16(11):931–40. Available from:
Soc [Internet]. 1914 Jun;36(6):1103–34. Available from:
[19] Rustum AM, Hoffman NE. Determination of cyclophosphamide in whole blood and plasma by reversed-phase high-performance
liquid chromatography. J Chromatogr B Biomed Sci Appl [Internet]. 1987 Jan;422:125–34. Available from: http://linkinghub.
[20] Rustum AM. Determination of cadralazine in human whole blood using reversed-phase high-performance liquid chromatography:
utilizing a salting-out extraction procedure. J Chromatogr [Internet]. 1989 Apr 14;489(2):345–52. Available from: http://www.
[21] Rustum AM. Determination of diltiazem in human whole blood and plasma by high-performance liquid chromatography
using a polymeric reversed-phase column and utilizing a salting-out extraction procedure. J Chromatogr [Internet]. 1989 May
30;490(2):365–75. Available from:
[22] Rustum AM. Estimation of cyclosporin-A in whole blood by simple and rapid reversed-phase HPLC utilizing a salting-out
extraction procedure. J Chromatogr Sci [Internet]. 1990 Nov;28(11):594–8. Available from:
[23] Cociglio M, Alric R, Bouvier O. Performance analysis of a reversed-phase liquid chromatographic assay of lamotrigine in plasma
using solvent-demixing extraction. J Chromatogr [Internet]. 1991 Dec 6;572(1-2):269–76. Available from: http://www.ncbi.nlm.
[24] Yoshida M, Akane A, Nishikawa M, Watabiki T, Tsuchihashi H. Extraction of Thiamylal in Serum Using Hydrophilic Acetonitrile
with Subzero-Temperature and Salting-Out Methods. Anal Chem [Internet]. 2004 Aug;76(16):4672–5. Available from: http://
[25] Kole PL, Venkatesh G, Kotecha J, Sheshala R. Recent advances in sample preparation techniques for effective bioanalytical
methods. Biomed Chromatogr [Internet]. 2011 Jan;25(1-2):199–217. Available from:
[26] Ramos RM, Valente IM, Rodrigues JA. Analysis of biogenic amines in wines by salting-out assisted liquid–liquid extraction and
high-performance liquid chromatography with fluorimetric detection. Talanta [Internet]. 2014 Jun;124:146–51. Available from:
[27] Gupta M, Jain A, Verma KK. Salt-assisted liquid–liquid microextraction with water-miscible organic solvents for the determination
of carbonyl compounds by high-performance liquid chromatography. Talanta [Internet]. 2009 Dec 15;80(2):526–31. Available
[28] Heydari R, Zarabi S. Development of combined salt- and air-assisted liquid–liquid microextraction as a novel sample preparation
technique. Anal Methods [Internet]. 2014 Sep 11;6(21):8469–75. Available from:
[29] Valente IM, Ramos RM, Gonçalves LM, Rodrigues JA. Determination of ethyl carbamate in spirits using salting-out assisted
liquid–liquid extraction and high performance liquid chromatography with fluorimetric detection. Anal Methods [Internet]. 2014
Sep 18;6(22):9136–41. Available from:
[30] Koltsakidou A, Zacharis CK, Fytianos K. A validated liquid chromatographic method for the determination of polycyclic aromatic
hydrocarbons in honey after homogeneous liquid–liquid extraction using hydrophilic acetonitrile and sodium chloride as mass
separating agent. J Chromatogr A [Internet]. 2015 Jan;1377:46–54. Available from:
[31] Gupta M, Pillai AKKV, Singh A, Jain A, Verma KK. Salt-assisted liquid–liquid microextraction for the determination of iodine
in table salt by high-performance liquid chromatography-diode array detection. Food Chem [Internet]. 2011 Feb;124(4):1741–6.
Available from:
[32] Røen BT, Sellevåg SR, Lundanes E. Quantification of Nerve Agent Biomarkers in Human Serum and Urine. Anal Chem
[Internet]. 2014 Dec 2;86(23):11833–40. Available from:
[33] Wang H, Gao M, Wang M, Zhang R, Wang W, Dahlgren RA, et al. Integration of phase separation with ultrasound-assisted
salt-induced liquid–liquid microextraction for analyzing the fluoroquinones in human body fluids by liquid chromatography. J
Chromatogr B [Internet]. 2015 Mar;985:62–70. Available from:
[34] Gao M, Wang H, Ma M, Zhang Y, Yin X, Dahlgren RA, et al. Optimization of a phase separation based magnetic-stirring
salt-induced liquid–liquid microextraction method for determination of fluoroquinolones in food. Food Chem [Internet]. 2015
May;175:181–8. Available from:
[35] TABATA M, KUMAMOTO M, NISHIMOTO J. Chemical Properties of Water-Miscible Solvents Separated by Salting-out
and Their Application to Solvent Extraction. Anal Sci [Internet]. 1994;10(3):383–8. Available from:
[36] Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ. Fast and easy multiresidue method employing acetonitrile extraction/
partitioning and “dispersive solid-phase extraction” for the determination of pesticide residues in produce. J AOAC Int [Internet].
86(2):412–31. Available from:
[37] Jain D, Athawale R, Bajaj A, Shrikhande S. Double-salting out assisted liquid–liquid extraction (SALLE) HPLC method for
estimation of temozolomide from biological samples. J Chromatogr B [Internet]. 2014 Nov;970:86–94. Available from: http://
[38] Polson C, Sarkar P, Incledon B, Raguvaran V, Grant R. Optimization of protein precipitation based upon effectiveness of protein
removal and ionization effect in liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life
Sci [Internet]. 2003 Mar 5;785(2):263–75. Available from:
[39] LI C-H. Recovery of Aniline from Wastewater by Nitrobenzene Extraction Enhanced with Salting-Out Effect. Biomed Environ
Sci [Internet]. 2010 Jun;23(3):208–12. Available from:
[40] Jenkins TF, Miyares PH. Nonevaporative preconcentration technique for volatile and semivolatile solutes in certain polar solvents.
Anal Chem [Internet]. 1991 Jul;63(13):1341–3. Available from:
[41] Tsai W-H, Huang T-C, Chen H-H, Wu Y-W, Huang J-J, Chuang H-Y. Determination of sulfonamides in swine muscle after
salting-out assisted liquid extraction with acetonitrile coupled with back-extraction by a water/acetonitrile/dichloromethane
ternary component system prior to high-performance liquid chromatography. J Chromatogr A [Internet]. 2010 Jan;1217(3):250–
5. Available from:
[42] Song S, Ediage EN, Wu A, De Saeger S. Development and application of salting-out assisted liquid/liquid extraction for multimycotoxin biomarkers analysis in pig urine with high performance liquid chromatography/tandem mass spectrometry. J Chromatogr
A [Internet]. 2013 May;1292:111–20. Available from:
[43] Zhao F-J, Tang H, Zhang Q-H, Yang J, Davey AK, Wang J. Salting-out homogeneous liquid–liquid extraction approach applied
in sample pre-processing for the quantitative determination of entecavir in human plasma by LC–MS. J Chromatogr B [Internet].
2012 Jan;881-882:119–25. Available from:
[44] Hamed AM, Arroyo-Manzanares N, García-Campaña AM, Gámiz-Gracia L. Determination of Fusarium toxins in functional
vegetable milks applying salting-out-assisted liquid–liquid extraction combined with ultra-high-performance liquid
chromatography tandem mass spectrometry. Food Addit Contam Part A [Internet]. 2017 Nov 2;34(11):2033–41. Available from:
[45] Tandel D, Shah P, Patel K, Thakkar V, Patel K, Gandhi T. Salting-Out Assisted Liquid–Liquid Extraction for Quantification
of Febuxostat in Plasma Using RP-HPLC and Its Pharmacokinetic Application. J Chromatogr Sci [Internet]. 2016 Sep
6;54(10):1827–33. Available from:
[46] Hendriks G, Uges DRA, Franke JP. Reconsideration of sample pH adjustment in bioanalytical liquid–liquid extraction of
ionisable compounds. J Chromatogr B [Internet]. 2007 Jun 15;853(1-2):234–41. Available from:
[47] Wang M, Cai Z, Xu L. Coupling of acetonitrile deproteinization and salting-out extraction with acetonitrile stacking in chiral
capillary electrophoresis for the determination of warfarin enantiomers. J Chromatogr A [Internet]. 2011 Jul;1218(26):4045–51.
Available from:
[48] Magiera S, Kolanowska A, Baranowski J. Salting-out assisted extraction method coupled with hydrophilic interaction liquid
chromatography for determination of selected β-blockers and their metabolites in human urine. J Chromatogr B [Internet]. 2016
Jun;1022:93–101. Available from:
[49] Fan Y, Hu S, Liu S. Salting-out assisted liquid-liquid extraction coupled to dispersive liquid-liquid microextraction for the
determination of chlorophenols in wine by high-performance liquid chromatography. J Sep Sci [Internet]. 2014 Dec;37(24):3662–
8. Available from:
[50] Atlabachew M, Chandravanshi BS, Redi-Abshiro M. Preparative HPLC for large scale isolation, and salting-out assisted liquid–
liquid extraction based method for HPLC–DAD determination of khat (Catha edulis Forsk) alkaloids. Chem Cent J [Internet].
2017 Dec 17;11(1):107. Available from:
[51] Liu G, Zhou N, Zhang M, Li S, Tian Q, Chen J, et al. Hydrophobic solvent induced phase transition extraction to extract
drugs from plasma for high performance liquid chromatography–mass spectrometric analysis. J Chromatogr A [Internet]. 2010
Jan;1217(3):243–9. Available from:
[52] Alshishani A, Salhimi SM, Saad B. Salting-out assisted liquid-liquid extraction coupled with hydrophilic interaction
chromatography for the determination of biguanides in biological and environmental samples. J Chromatogr B [Internet]. 2018
Jan;1073:51–9. Available from:
[53] Tan B, Negahban A, McDonald T, Zhang Y, Holliman C. Utilization of hydrophilic-interaction LC to minimize matrix effects
caused by phospholipids. Bioanalysis [Internet]. 2012 Aug;4(16):2049–58. Available from:
[54] Li T, Zhang L, Tong L, Liao Q. High-throughput salting-out-assisted homogeneous liquid-liquid extraction with acetonitrile
for determination of baicalin in rat plasma with high-performance liquid chromatography. Biomed Chromatogr [Internet]. 2014
May;28(5):648–53. Available from:
[55] Xiong X, Zhang L, Cheng L, Mao W. High-throughput salting-out assisted liquid-liquid extraction with acetonitrile for the
determination of anandamide in plasma of hemodialysis patients with liquid chromatography tandem mass spectrometry. Biomed
Chromatogr [Internet]. 2015 Sep;29(9):1317–24. Available from:
[56] Liu Z, Yu W, Zhang H, Gu F, Jin X. Salting-out homogenous extraction followed by ionic liquid/ionic liquid liquid–liquid microextraction for determination of sulfonamides in blood by high performance liquid chromatography. Talanta [Internet]. 2016
Dec;161:748–54. Available from:
[57] Niu Z, Yu C, He X, Zhang J, Wen Y. Salting-out assisted liquid–liquid extraction combined with gas chromatography-mass
spectrometry for the determination of pyrethroid insecticides in high salinity and biological samples. J Pharm Biomed Anal
[Internet]. 2017 Sep;143:222–7. Available from:
[58] Tsai C-J, Liao F-Y, Weng J-R, Feng C-H. Tandem derivatization combined with salting-out assisted liquid–liquid microextraction
for determination of biothiols in urine by gas chromatography–mass spectrometry. J Chromatogr A [Internet]. 2017 Nov;1524:29–
36. Available from: