Capillary electrophoresis-mass spectrometry of Spirulina platensis proteins obtained by pressurized liquid extraction

In this work, the usefulness of CE-MS to monitor and optimize the pressurized liquid extraction (PLE) of proteins from Spirulina platensis microalga is demonstrated. Crude and purified PLE extracts from microalga were analyzed by CE-MS. It was observed that the use of purification protocols of phycobiliproteins (namely, ultrafiltration or precipitation-dialysis-freeze drying) resulted in better CE resolution and MS signals, demonstrating that sample matrix plays an important role in CE-MS of proteins in real samples. Ultrafiltration was found less laborious and much faster than precipitation-dialysis-freeze drying (1 vs. 48 h). Direct analysis of crude extracts was demonstrated to be also possible by CE-MS, providing less-quality information but enough to characterize PLE extracts in a much faster way. Therefore, the latter protocol was selected to monitor and optimize the extraction process of phycobiliproteins from S. platensis. To do that, different extraction conditions were tested, including time, temperature and pressure of extraction, nature of pressurized liquid, distribution of microalga inside the extraction cell, type of packing, etc. It is demonstrated that the combined use of PLE and CE-MS allows the attainment of extracts rich in phycobiliproteins in short extraction times (namely, yields of 20% can be obtained in less than 2 h under the optimum PLE process in an automatic way). To our knowledge, this work shows for the first time the usefulness of CE-MS for monitoring and optimizing a PLE process.     

Development of a pressurized liquid extraction and clean-up procedure for the determination of alpha-endosulfan, beta-endosulfan and endosulfan sulfate in aged contaminated Ethiopian soils

Pressurized liquid extraction (PLE) was investigated for the extraction of two endosulfan isomers and their metabolite from two real contaminated soil samples. PLE for 3x10min at 100 degrees C was proven to be more exhaustive than Soxhlet extraction (SOX) in one soil sample. On the other soil sample investigated the method was found to be equally exhaustive as SOX. The use of hazardous organic solvents such as n-hexane, toluene, and diethyl ether has been avoided in PLE and clean-up. Instead less toxic solvents have been used both at the extraction step (acetone/n-heptane) and clean-up step (ethyl acetate/n-heptane). A column Florisil clean-up procedure that consumes relatively low solvent volumes has been optimized and applied to purify soil extracts. The developed analytical procedure was validated by applying it to a certified reference soil material (CRM811-050). A recovery of 103% total endosulfan residue was obtained versus certified values.     

Selective pressurized liquid extraction for multi-residue analysis of organochlorine pesticides in soil

A selective pressurized liquid extraction (SPLE) procedure capable of performing simultaneous extraction and clean-up has been demonstrated for multi-residue analysis of organochlorine pesticides (OCPs) in soil. The final method was performed at 100 degrees C for 3 x 10 min using acetone/n-heptane (1:1, v/v). Florisil was placed inside the extraction cell downstream the sample to remove interfering compounds. Extraction of two soil samples by SPLE gave a recovery which was over 80% for beta-endosulfan, endosulfan sulfate, p,p'-DDT and p,p'-DDE compared to PLE with off-line clean-up. The same trend was observed when applying the SPLE method to a certified reference soil sample (CRM 811-050) containing 13 OCPs, where the SPLE method gave 80-90% recovery vis-à-vis the PLE method with off-line clean-up. Feasibility of the SPLE method was further demonstrated by applying it to five real soil samples collected in Ethiopia.     

Determination of gastrodin and vanillyl alcohol in Gastrodia elata Blume by pressurized liquid extraction at room temperature

Pressurized liquid extraction (PLE) at room temperature with a laboratory-assembled system was applied for the extraction of gastrodin (GA) and vanillyl alcohol (VA) in Gastrodia elata Blume. The proposed system setup for this current work was simpler as no heating and backpressure regulator was required. Extraction with PLE was carried out dynamically at a flow rate of 1.5 mL/min, at room temperature, under an applied pressure of 10-20 bars with an extraction time of 40-50 min. The extraction efficiencies of the proposed method using 20% aqueous ethanol were compared with heating under reflux using organic solvents such as methanol and ethanol/water (20:80) for different batches of medicinal plant materials. For the determination of GA and VA in G. elata Blume, the extraction efficiencies of PLE at room temperature were observed to be comparable with heating under reflux. The method precision was found to vary from 1.6 to 8.6% (RSD, n = 6) on different days. The marker compounds present in the various medicinal plant extracts were determined by gradient elution HPLC and HPLC/MS/MS. Our work demonstrated the possibility of implementation of PLE at room temperature and the advantages of minimizing the use of organic solvents in the extraction process.     

Miniaturised selective pressurised liquid extraction of polychlorinated biphenyls from foodstuffs

The feasibility of miniaturised pressurised liquid extraction (PLE) with in-cell purification and subsequent gas chromatography with micro-electron capture detection (GC-micro-ECD) for the determination of prioritary and toxic polychlorinated biphenyls (PCBs) in a variety of foodstuffs (fat contents in the range 22-49%, w/w, on a freeze-dried basis) has been investigated. After optimisation of the several experimental parameters affecting the efficiency of the selective PLE process, the developed method provided quantitative recoveries of the endogenous PCBs studied and complete fat elimination in a single step using n-hexane as extraction solvent. A total solvent volume of 3.5 mL was used for the two consecutive 7 min static PLEs of 100-mg samples. Detection limits using GC-micro-ECD were below 0.2 ng/g freeze dried sample for all 22 PCBs investigated in real-life foodstuffs, and the repeatability of the complete PLE plus GC-micro-ECD method as calculated for the analysis of the endogenous PCBs in general was better than 14%. Comparison of the miniaturised PLE method developed with either conventional Soxhlet extraction or matrix solid phase dispersion with subsequent (off-line) clean-up for the analysis of non-spiked samples showed that the efficiency of PLE was similar to or better (recoveries in the range 83-133%, as calculated for the endogenous analytes) than for the other two extraction methods assayed.     

Miniaturised pressurised liquid extraction of polycyclic aromatic hydrocarbons from soil and sediment with subsequent large-volume injection-gas chromatography

Analyte extraction is the main limitation when developing at-line, or on-line, procedures for the preparation of (semi)solid environmental samples. Pressurised liquid extraction (PLE) is an analyte- and matrix-independent technique which provides cleaner extracts than the time-consuming classical procedures. In the study, the practicality of miniaturised PLE performed in a stainless-steel cell, and combined with subsequent large-volume injection (LVI)-GC-MS was studied. As an example, the new system was applied to the determination of polycyclic aromatic hydrocarbons (PAHs) in soils and a sediment. Variables affecting the PLE efficiency, such as pressure and temperature of the extraction solvent and total solvent volume, were studied. Toluene was selected as extraction solvent and a total solvent volume of 100 microl was used for the 10 min static-dynamic PLE of 50-mg samples. Additional clean-up or filtration of the sample extracts was not required. Detection limits using LVI-GC-MS were below 9 ng/g soil for the 13 PAHs more volatile than indeno[1,2,3-cd]pyrene in real soil samples and the repeatability of the complete PLE plus LVI-GC-MS method for the analysis of the endogenous PAH was better than 15%. Comparison of PLE and Soxhlet or liquid-partitioning extraction results for the analysis of non-spiked samples showed that the efficiency of PLE is the same or better than for the other two extraction methods assayed.     

Analytical method for the determination of trace levels of steroid hormones and corticosteroids in soil, based on PLE/SPE/LC-MS/MS

The aim of this study was to develop an efficient, sensitive and reliable analytical method for the determination of traces of steroid hormones (including oestrogen, androgens and progestagens) and corticosteroids in soil. A method of sample preparation involving pressurized liquid extraction (PLE) and solid-phase extraction (SPE) was developed for the determination of six steroids and five corticosteroids in soils, followed by analysis by liquid chromatography-tandem mass spectrometry. The conditions employed for PLE involved acetone/methanol (50:50) as the extracting solvent, a temperature of 80 °C, two cycles and a static time of 5 min. The extraction was followed by a SPE clean-up based on a polymeric phase. With use of protocol, a residual matrix effect was, however, highlighted. The limit of detection in soil was 0.08–0.89 ng/g for steroids and 0.09–2.84 ng/g for corticosteroids.     

Pressurized liquid extraction of polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and coplanar polychlorinated biphenyls from contaminated soil

Extraction solvents for pressurized liquid extraction (PLE) used to extract polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans (PCDD/PCDFs), and coplanar polychlorinated biphenyls (Co-PCBs) from contaminated soil were investigated. The PCDD/PCDFs and Co-PCBs in Certified Reference Material: CRM 0422 (Forest soil) were extracted using toluene, n-hexane, acetone, acetone/toluene and acetone/n-hexane (1:1, v/v). Soxhlet extraction was the reference method. Results demonstrated that PLE using mixed solvents produced better analyte recoveries than the single solvents. However, these results were lower than those for Soxhlet extraction. Additional extraction cycles using mixed solvents achieved better recovery results. Mixed solvents and several extraction cycles were necessary for satisfactory extraction of more tightly bound PCDD/PCDFs and Co-PCBs from soil.     

Suitability of selective pressurized liquid extraction combined with gas chromatography-ion-trap tandem mass spectrometry for the analysis of polybrominated diphenyl ethers

A one-step extraction and clean-up method using pressurized liquid extraction (PLE) (selective PLE) combined with gas chromatography-ion-trap tandem mass spectrometry (GC-ITMS-MS) was evaluated for the analysis of polybrominated diphenyl ethers (from tri- to hepta-PBDEs) at low concentrations in fish and shellfish samples. To this end, the performance of an on-line PLE extraction/clean-up method and of a classical Soxhlet extraction and clean-up method using a multi-layer modified silica column were compared. The two sample treatment methods provided similar results, although an important reduction in the sample treatment time (40 min per sample) was achieved using the selective PLE method. In addition, the suitability of the PLE combined with GC-ITMS-MS method was evaluated by comparing the results obtained in the analysis of fish samples with those obtained by gas chromatography-high resolution mass spectrometry (GC-HRMS). Good agreement between both techniques was obtained with differences between the mean values of less than 16%. The selective PLE method coupled to GC-ITMS-MS produced accurate results for PBDE determination with low limits of detection (1.0-16.8 pg g⁻¹ wet weight) and quantification (3.1-51 pg g⁻¹ wet weight) as well as good precision (RSD < 16%). This method has been applied to the analysis of PBDEs in fish and shellfish samples collected at fish markets in Catalonia (NE Spain).     

Optimisation of pressurised liquid extraction for the determination of p,p′-DDT and p,p′-DDE in aged contaminated Ethiopian soils

The effectiveness of extracting p,p′-DDT and p,p′-DDE from aged contaminated soil samples by means of pressurized liquid extraction (PLE) was evaluated. Two soil samples, which were contaminated more than 10 years ago, were used in the investigation. The static extraction time was optimised and then validated against the total sum of target analytes obtained from multiple sequential extractions. The PLE results were also compared with Soxhlet extraction (SOX). PLE for 3×10 min at 100 °C was proven to be more exhaustive than SOX in the determination of p,p′-DDE from both soil samples. In the case of p,p′-DDT, PLE was found to be equally as exhaustive as SOX. Additionally, most of the previous PLE investigations used hazardous organic solvents such as n-hexane, toluene and dichloromethane mixed with acetone, whereas in this investigation the less toxic solvent combination n-heptane/acetone has been employed.     

Determination of bisphenol diglycidyl ether residues in canned foods by pressurized liquid extraction and liquid chromatography-tandem mass spectrometry

A new confirmatory method for simultaneous determination of bisphenol diglycidyl ether residues (BADGE, BADGE.H(2)O, BADGE.2H(2)O, BADGE.H(2)O.HCl, BADGE.HCl, BADGE.2HCl, BFDGE and BFDGE.2HCl) from canned food has been developed. The proposed method includes extraction by pressurized liquid extraction (PLE) followed by liquid-liquid partition and purification by solid phase extraction (SPE). Several solvent systems and different operating conditions (time, temperature) have been investigated for PLE optimization. A reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to atmospheric pressure chemical ionisation tandem mass spectrometry (APCI-MS-MS) method was developed for the separation, quantification and confirmation. The ion source settings were optimized using a design of experiments (DOE). The optimized method was applied to the determination of these chemicals at very low levels in different samples with a quantification limit of 5 ng/g. Recoveries ranged between 82 and 101% and standard deviations were less than 10%.     

Analysis of insecticides in honey by liquid chromatography-ion trap-mass spectrometry: comparison of different extraction procedures

The feasibility of different extraction procedures was tested and compared for the determination of 12 organophosphorus and carbamates insecticides in honey samples. In this sense, once the samples were pre-treated - essentially dissolved in hot water by stirring - and before they could be analyzed by liquid chromatography-ion trap-second stage mass spectrometry (LC-MS(2)), four different approaches were studied for the extraction step: QuEChERS, solid-phase extraction (SPE), pressurized liquid extraction (PLE) and solid-phase microextraction (SPME). The main aim of this work was to maximise the sensitivity of pesticides and to minimise the presence of interfering compounds in the extract. All pesticides were linear in the range from CC(β) to 1000× CC(β) for the four extraction methods (three orders of magnitude). Detection capabilities (CC(β)) were 0.024-1.155 mg kg(-1) with QuEChERS, 0.010-0.646 mg kg(-1) with SPE, 0.007-0.595 mg kg(-1) with PLE, and 0.001-0.060 mg kg(-1) with SPME. All the target compounds could be recovered by any of the methods, at a CC(β) fortification level ranged from 28 to 90% for the SPME. In comparison, the PLE method was the most efficient extraction method with recoveries from 82 to 104%. It was followed by the QuEChERS method with recoveries between 78 and 101% and the SPE method with recoveries between 72 and 100%. The repeatability expressed as relative standard deviation (RSDs) was below 20% for all the pesticides by any of the tested extraction methods. Results obtained applying the four extraction techniques to real honey samples are analogous.     

New possibilities for the valorization of olive oil by-products

In this contribution, the capabilities of pressurized liquid extraction (PLE) using food-grade solvents, such as water and ethanol, to obtain antioxidant extracts rich on polyphenolic compounds from olive leaves are studied. Different extraction conditions were tested, and the PLE obtained extracts were characterized in vitro according to their antioxidant capacity (using the DPPH radical scavenging and the TEAC assays) and total phenols amounts. The most active extracts were obtained with hot pressurized water at 200 °C (EC(50) 18.6 μg/mL) and liquid ethanol at 150 °C (EC(50) 27.4 μg/mL), attaining at these conditions high extraction yields, around 40 and 30%, respectively. The particular phenolic composition of the obtained extracts was characterized by LC-ESI-MS. Using this method, 25 different phenolic compounds could be tentatively identified, including phenolic acids, secoiridoids, hydroxycinnamic acid derivatives, flavonols and flavones. Among them, hydroxytyrosol, oleuropein and luteolin-glucoside were the main phenolic antioxidants and were quantified on the extracts together with other minor constituents, by means of a UPLC-MS/MS method. Results showed that using water as extracting agent, the amount of phenolic compounds increased with the extraction temperature, being hydroxytyrosol the main phenolic component on the water PLE olive leaves extracts, reaching up to 8.542 mg/g dried extract. On the other hand, oleuropein was the main component on the extracts obtained with ethanol (6.156-2.819 mg/g extract). Results described in this work demonstrate the good possibilities of using PLE as a useful technique for the valorization of by-products from the olive oil industry, such as olive leaves.     

Comparisons of soxhlet extraction, pressurized liquid extraction, supercritical fluid extraction and subcritical water extraction for environmental solids: recovery, selectivity and effects on sample matrix

Extractions of a polycyclic aromatic hydrocarbon (PAH)-contaminated soil from a former manufactured gas plant site were performed with a Soxhlet apparatus (18 h), by pressurized liquid extraction (PLE) (50 min at 100 degrees C), supercritical fluid extraction (SFE) (1 h at 150 degrees C with pure CO2), and subcritical water (1 h at 250 degrees C, or 30 min at 300 degrees C). Although minor differences in recoveries for some PAHs resulted from the different methods, quantitative agreement between all of the methods was generally good. However, the extract quality differed greatly. The organic solvent extracts (Soxhlet and PLE) were much darker, while the extracts from subcritical water (collected in toluene) were orange, and the extracts from SFE (collected in CH2Cl2) were light yellow. The organic solvent extracts also yielded more artifact peaks in the gas chromatography (GC)-mass spectrometry and GC-flame ionization detection chromatograms, especially compared to supercritical CO2. Based on elemental analysis (carbon and nitrogen) of the soil residues after each extraction, subcritical water, PLE, and Soxhlet extraction had poor selectivity for PAHs versus bulk soil organic matter (approximately 1/4 to 1/3 of the bulk soil organic matter was extracted along with the PAHs), while SFE with pure CO2 removed only 8% of the bulk organic matrix. Selectivities for different compound classes also vary with extraction method. Extraction of urban air particulate matter with organic solvents yields very high concentrations of n- and branched alkanes (approximately C18 to C30) from diesel exhaust as well as lower levels of PAHs, and no selectivity between the bulk alkanes and PAHs is obtained during organic solvent extraction. Some moderate selectivity with supercritical CO2 can be achieved by first extracting the bulk alkanes at mild conditions, followed by stronger conditions to extract the remaining PAHs, i.e., the least polar organics are the easiest organics to extract with pure CO2. In direct contrast, subcritical water prefers the more polar analytes, i.e., PAHs were efficiently extracted from urban air particulates at 250 degrees C, with little or no extraction of the alkanes. Finally, recent work has demonstrated that many pollutant molecules become "sequestered" as they age for decades in the environment (i.e., more tightly bound to soil particles and less available to organisms or transport). Therefore, it may be more important for an extraction method to only recover pollutant molecules that are environmentally-relevant, rather than the conventional attempts to extract all pollutant molecules regardless of how tightly bound they are to the soil or sediment matrix. Initial work comparing SFE extraction behavior using mild to strong conditions with bioremediation behavior of PAHs shows great promise to develop extraction methodology to measure environmentally-relevant concentrations of pollutants in addition to their total concentrations.     

Optimizing pressurized liquid extraction of microbial lipids using the response surface method

Response surface methodology (RSM) was used for the determination of optimum extraction parameters to reach maximum lipid extraction yield with yeast. Total lipids were extracted from oleaginous yeast (Rhodotorula glutinis) using pressurized liquid extraction (PLE). The effects of extraction parameters on lipid extraction yield were studied by employing a second-order central composite design. The optimal condition was obtained as three cycles of 15 min at 100°C with a ratio of 144 g of hydromatrix per 100 g of dry cell weight. Different analysis methods were used to compare the optimized PLE method with two conventional methods (Soxhlet and modification of Bligh and Dyer methods) under efficiency, selectivity and reproducibility criteria thanks to gravimetric analysis, GC with flame ionization detector, High Performance Liquid Chromatography linked to Evaporative Light Scattering Detector (HPLC-ELSD) and thin-layer chromatographic analysis. For each sample, the lipid extraction yield with optimized PLE was higher than those obtained with referenced methods (Soxhlet and Bligh and Dyer methods with, respectively, a recovery of 78% and 85% compared to PLE method). Moreover, the use of PLE led to major advantages such as an analysis time reduction by a factor of 10 and solvent quantity reduction by 70%, compared with traditional extraction methods.     

Extraction of rotenone from Derris elliptica and Derris malaccensis by pressurized liquid extraction compared with maceration

The extraction of active compounds from plants is one of the most critical steps in the commercial development of natural products for medicinal, herbicidal or pesticidal use. The focus of this study was to compare conventional maceration and pressurized liquid extraction (PLE) techniques for the efficient extraction of rotenone from the stem and root of Derris elliptica Benth and Derris malaccensis Prain. The effects of experimental variables, such as solvent, temperature and pressure, on PLE efficiency have been studied. Chloroform was determined to be a good extraction solvent (rotenone content 40.6%, w/w) compared to commonly used solvent, 95% ethanol (rotenone content 15.0%, w/w). The optimal conditions for PLE were 50 degrees C and 2000 psi. PLE showed higher extraction efficiency (rotenone content 46.1%, w/w) as compared with conventional maceration method (rotenone content 40.6%, w/w). The order of rotenone content found in crude extract obtained by optimized method from the highest to the lowest was root (46.1%, w/w) and stem (9.4%, w/w) of D. elliptica and stem of D. malaccensis (5.2%, w/w), respectively. Moreover, the results from this study indicated that PLE was considerably less time and solvent consuming (30 min, 3 ml/g of dried sample) than the conventional maceration techniques (72 h, 10 ml/g of dried sample).     

Development of a pressurised liquid extraction and liquid chromatography with electrospray ionization-tandem mass spectrometry method for the determination of domoic acid in shellfish

Amnesic shellfish poisoning (ASP) is a potentially lethal human toxic syndrome which is caused by domoic acid (DA) that originates in marine phytoplankton belonging to the Pseudonitzschia genus. A confirmatory and sensitive procedure has been developed and validated for the determination of DA in shellfish. The proposed method includes pressurised liquid extraction (PLE) with methanol/acetone (9:1), florisil clean-up purification inside the PLE extraction cell and detection by liquid chromatography (LC) coupled to electrospray ionization in positive mode tandem mass spectrometry (ESI-MS-MS). Comparison of ionization sources (ESI, atmospheric pressure ionization (APCI) atmospheric pressure photoionization (APPI) and combined APCI/APPI) were carried out in order to improve the analytical signal. The main parameters affecting the performance of the different ionization sources and PLE parameters were previously optimised using statistical design of experiments (DOE). Linear calibrations were obtained using mussel tissue extracts 0.05-5 microg DA/ml (R2>0.999). The limits of detection (LOD) and quantitation (LOQ) of the method were 0.2 and 0.5 microg/g respectively and recoveries ranged from 81 to 95%. This method was successfully applied to determine DA levels in 46 shellfish samples collected from Valencian (Spain) supermarkets, showing high sample throughput.     

Comparison of ultrasonic and pressurized liquid extraction for the analysis of polycyclic aromatic compounds in soil samples by gas chromatography coupled to tandem mass spectrometry

A pressurized liquid extraction (PLE) method has been optimized for the determination of polycyclic aromatic hydrocarbons (PAHs) in soil samples and it was compared with ultrasonic extraction. The extraction step was followed by gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS/MS) analysis. Parameters such as type of solvent, extraction time, extraction temperature and number of extractions were optimized. There were no significant differences among the two extraction methods although better extraction efficiencies were obtained when PLE was used, minimizing extraction time and solvent consumption. PLE procedure was validated, obtaining limits of detection (LODs) ranging from 0.02 to 0.75 μg kg⁻¹ and limits of quantification (LOQs) ranging from 0.07 to 2.50 μg kg⁻¹ for the selected PAHs. Recoveries were in the range of 59-110%, except for naphthalene, which was the most volatile PAH. Finally, the method was applied to real soil samples from Southeast of Spain. PAHs concentrations were low, and phenanthrene, pyrene, fluorene, benzo[a]pyrene and chrysene were the most frequently detected analytes in the samples.     

Simultaneous extraction of polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and coplanar polychlorinated biphenyls from contaminated soil using pressurized liquid extraction

Pressurized liquid extraction (PLE) was studied for simultaneous extractions of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDFs), and of coplanar polychlorinated biphenyls (Co-PCBs) from a tightly bounded condition in the soil matrix. Temperatures were maintained at 175 and 200 degrees C, respectively, and two or three static cycles for single PLE with toluene and acetone/n-hexane were studied using a certified reference material to compare the effects of those conditions on recoveries. A single PLE with two static cycles at 150 degrees C and the repeated single PLE (additional PLE) were reference methods. Satisfactory recoveries were not obtained using tested single PLE (2,3,7,8-substituted PCDD/PCDFs and Co-PCBs average, average (avg.) 79-103%), but they were achieved using additional PLE (acetone/n-hexane, avg. 115-128%; toluene, avg. 111-132%). In addition, these methods and additional PLE of the reference method using acetone/n-hexane were not markedly different (avg. 123-128%). That fact suggests that the use of mixed solvents and additional PLE were more important factors than temperatures and static cycles of single PLE for quantitative and simultaneous extractions of those compounds from the soil.     

Determination of N-nitroso derivatives of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in soils by pressurized liquid extraction and liquid chromatography-electrospray ionization mass spectrometry

To aid in the evaluation of the potential toxicity of N-nitroso derivatives of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), we describe a pressurized liquid extraction (PLE) followed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method for determination of RDX and its N-nitroso derivatives: hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) in soils. Sandy loam soil was spiked with RDX and its N-nitroso derivatives (MNX, DNX, and TNX). Acetonitrile was used as the PLE extraction solvent at 100 degrees C and 1500 psi for 15 min. Florisil was used to cleanup extracts following PLE. Instrumental analysis employed LC-ESI-MS, in which 1mM acetic acid was added to the mobile phase to facilitate formation of acetate adduct ions [M+CH(3)COO](-). The method detection limits (MDLs) for RDX, MNX, DNX, and TNX were 1.46, 1.46, 1.69, and 1.93 ng/g, respectively. High recovery (91.1-108.3%), good precision (RSD: 3.2-12.4%), and reproducibility were achieved. This method proved effective and was applied to monitor the reductive biotransformation of MNX in soils with the presence of earthworms (Eisenia fetida).