## 4 Conclusions  
In conclusion, a simple, LLE-based sample preparation protocol for the metabolic profiling of urine samples was optimized and validated. The method was reliable and does not require specific instrumentation for sample preparation. We successfully demonstrated for the first time the applicability of single urine solvent extracts for both GC-MS as well as \({}^{1}\)H-NMR analysis, for volatile and semi-volatile compound analysis. The possibility of obtaining a wider range of the metabolites can give more information about the health of the individuals, and may facilitate the identification of biomarkers linked to different diseases. The possibility of using both GC-MS and \({}^{1}\)H-NMR platforms allowed the detection of metabolites with different volatilities from a single sample. This may be useful when carrying out future studies aimed at identifying metabolites linked to disease. Often, previous studies have had limited scope and concentrate on a limited range of methods. Using these methods, the detection capability could be increased many-fold by simply increasing the extraction volumes used; for instance, 40 mL DCM and 20 mL urine. The benefit of the proposed method is the possibility of storage of extracts in smaller vials, as compared to intact urine samples, and the possibility of transport to different labs for analysis in a dry way, without the special freezing conditions.  
Future work will involve the application of the optimized method to look for diagnostic markers of urological cancers. In summary, LLE has been shown to be superior to the SPME fiber pre-concentration methodology for urine analyses, permitting semi-volatiles to be detected and providing a promising alternative methodology to the use of very costly absorptive technologies.  
**Supplementary Materials:** The following are available online, Supplementary Table S1. Tentative assignment of metabolites found to be presented in urine aqueous phase (\({}^{1}\)H-NMR analyses). Supplementary Figure S1. The comparison of the extraction using dichloromethane (DCM), chloroform and diethyl ether. Supplementary Figure S2 \({}^{1}\)H-NMR 600 MHz CPMG spectra of urine obtained from polar phase sample (D2O, T=300K). **1--**2-hydroxisyovaleric acid, **2--**3-methyl-2-oxovaleric acid, **3--**sovaleric acid, **4--**Valine, **5--**3-hydroxyisobutyric acid, **6--**Methylsuccinic acid, **7--**Fucose, **8--**3-hydroxyisovaleric acid, **9--**2-**3-hydroxyisobutyric acid, **10--**2-**2-**phenylpropionic acid, **11--**Alanine, **12--**2-**2-aminoadipic acid, **13--**Acetate, **14--**Acetate, **15--**Acetate, **16--**Acetate acid, **17--**Sucic acid, **18--**Citrate, **19--**Sacrosine, **20--**Dimethylamine, **21--**Trimepluralime, **22--**3--**N_,_N_-dimethylbycine, **23--**N_-methylydivation, **24--**Creatine, **25--**Creatinine, **26--**Choline, **27--**Methanol, **28--**Glycine, **29--**Glycolate, 30--**7--**methylistidine, **31--**Trigonline, **32--**Fumaric acid, **33--**trans-Aconitic acid, **34--**Xanthuretic acid, **35--**Carnosine, **36--**3-**Indoxylsulfate, **37--**Imidazole, **38--**Hippurate, **39--**Oxypurinol, **40--**Adenine, **41--**Formic acid, **42--**1-**methylnicotinamide.  
**Author Contributions: [PERSON], [PERSON] and [PERSON] conceived and designed the research; B.d.L.C. secured funding; [PERSON], [PERSON], [PERSON] and [PERSON] conducted analytical experiments; [PERSON] analyzed GC-MS data; [PERSON], [PERSON] and [PERSON] analysed \({}^{1}\)H NMR data; [PERSON], [PERSON] and [PERSON] interpreted results; [PERSON] wrote the draft of the manuscript; [PERSON], [PERSON], [PERSON], [PERSON] and [PERSON] contributed in the further writing of the manuscript. All authors read and approved the final version of the manuscript.**  
**Funding: The study was supported by Project No. GA2543 from the Above and Beyond Charity in Bristol.**  
**Acknowledgments: [PERSON] is a START 2019 scholarship holder financed by the Foundation for Polish Science (FNP).**  
**Conflicts of Interest: The authors declare no conflict of interest.**