Affinity partitioning of plasmid DNA with a zinc finger protein

The affinity isolation of pre-purified plasmid DNA (pDNA) from model buffer solutions using native and poly(ethylene glycol) (PEG) derivatized zinc finger-GST (Glutathione-S-Transferase) fusion protein was examined in PEG-dextran (DEX) aqueous two-phase systems (ATPSs). In the absence of pDNA, partitioning of unbound PEGylated fusion protein into the PEG-rich phase was confirmed with 97.5% of the PEGylated fusion protein being detected in the PEG phase of a PEG 600-DEX 40 ATPS. This represents a 1322-fold increase in the protein partition coefficient in comparison to the non-PEGylated protein (Kc = 0.013). In the presence of pDNA containing a specific oligonucleotide recognition sequence, the zinc finger moiety of the PEGylated fusion protein bound to the plasmid and steered the complex to the PEG-rich phase. An increase in the proportion of pDNA that partitioned to the PEG-rich phase was observed as the concentration of PEGylated fusion protein was increased. Partitioning of the bound complex occurred to such an extent that no DNA was detected by the picogreen assay in the dextran phase. It was also possible to partition pDNA using a non-PEGylated (native) zinc finger-GST fusion protein in a PEG 1000-DEX 500 ATPS. In this case the native ligand accumulated mainly in the PEG phase. These results indicate good prospects for the design of new plasmid DNA purification methods using fusion proteins as affinity ligands.     

Purification of plasmid DNA vectors by aqueous two-phase extraction and hydrophobic interaction chromatography

The current study explores the possibility of using a polyethyleneglycol(PEG)-ammonium sulphate aqueous two-phase system (ATPS) as an early step in a process for the purification of a model 6.1 kbp plasmid DNA (pDNA) vector. Neutralised alkaline lysates were fed directly to ATPS. Conditions were selected to direct pDNA towards the salt-rich bottom phase, so that this stream could be subsequently processed by hydrophobic interaction chromatography (HIC). Screening of the best conditions for ATPS extraction was performed using three PEG molecular weights (300, 400 and 600) and varying the tie-line length, phase volume ratio and lysate load. For a 20% (w/w) lysate load, the best results were obtained with PEG 600 using the shortest tie-line (38.16%, w/w). By further manipulating the system composition along this tie-line in order to obtain a top/bottom phase volume ratio of 9.3 (35%, w/w PEG 600, 6%, w/w NH4)2 SO4), it was possible to recover 100% of pDNA in the bottom phase with a three-fold increase in concentration. Further increase in the lysate load up to 40% (w/w) with this system resulted in a eight-fold increase in pDNA concentration, but with a yield loss of 15%. The ATPS extraction was integrated with HIC and the overall process compared with a previously defined process that uses sequential precipitations with iso-propanol and ammonium sulphate prior to HIC. Although the final yield is lower in the ATPS-based process the purity grade of the final pDNA product is higher. This shows that it is possible to substitute the time-consuming two-step precipitation procedure by a simple ATPS extraction.     

Downstream processing of antibodies: Single-stage versus multi-stage aqueous two-phase extraction

Single-stage and multi-stage strategies have been evaluated and compared for the purification of human antibodies using liquid–liquid extraction in aqueous two-phase systems (ATPSs) composed of polyethylene glycol 3350 (PEG 3350), dextran, and triethylene glycol diglutaric acid (TEG-COOH). The performance of single-stage extraction systems was firstly investigated by studying the effect of pH, TEG-COOH concentration and volume ratio on the partitioning of the different components of a Chinese hamster ovary (CHO) cells supernatant. It was observed that lower pH values and high TEG-COOH concentrations favoured the selective extraction of human immunoglobulin G (IgG) to the PEG-rich phase. Higher recovery yields, purities and percentage of contaminants removal were always achieved in the presence of the ligand, TEG-COOH. The extraction of IgG could be enhanced using higher volume ratios, however with a significant decrease in both purity and percentage of contaminants removal. The best single-stage extraction conditions were achieved for an ATPS containing 1.3% (w/w) TEG-COOH with a volume ratio of 2.2, which allowed the recovery of 96% of IgG in the PEG-rich phase with a final IgG concentration of 0.21 mg/mL, a protein purity of 87% and a total purity of 43%. In order to enhance simultaneously both recovery yield and purity, a four stage cross-current operation was simulated and the corresponding liquid–liquid equilibrium (LLE) data determined. A predicted optimised scheme of a counter-current multi-stage aqueous two-phase extraction was hence described. IgG can be purified in the PEG-rich top phase with a final recovery yield of 95%, a final concentration of 1.04 mg/mL and a protein purity of 93%, if a PEG/dextran ATPS containing 1.3% (w/w) TEG-COOH, 5 stages and volume ratio of 0.4 are used. Moreover, according to the LLE data of all CHO cells supernatant components, it was possible to observe that most of the cells supernatant contaminants can be removed during this extraction step leading to a final total purity of about 85%.     

Pressurized liquid extraction with in-cell clean-up followed by gas chromatography-tandem mass spectrometry for the selective determination of parabens and triclosan in indoor dust

This work studied the possibility of using polyethyleimine (PEI) as an affinity ligand for the purification of plasmid DNA (pDNA) from alkaline lysates using aqueous two-phase systems (ATPSs). The goal was to find conditions under which this cationic polymer could steer the partition of pDNA to the phase where less impurities accumulate. In poly(ethylene glycol) (PEG)/ammonium sulphate systems, neither free nor PEGylated PEI (pPEI) were able to change the partition of pDNA. This is probably due to the high salt concentration present in these systems that impair the interaction between pDNA and PEI. In PEG 3350/dextran 110 systems, the desired effect could be observed but 0.2-0.5M ammonium sulphate had to be added to prevent the co-partition of RNA to the same phase. These results were used to develop a methodology to obtain polyplexes from alkaline lysates in a two-step ATPSs extraction process. In the first step, a PEG 600/ammonium sulphate system is used to remove most impurities to the top phase. The pDNA-containing bottom phase is then isolated and contacted with a second PEG 3350/dextran 110 system supplemented with a small amount of pPEI (0.2%). Plasmid yield was 100% and the final preparation had no RNA and only small amounts of contaminant protein. Additionally, pDNA was obtained in the form of 53nm-sized polyplexes which are likely to suit specific gene delivery applications.     

Exploitation of the coil-globule plasmid DNA transition induced by small changes in temperature, pH salt, and poly(ethylene glycol) compositions for directed partitioning in aqueous two-phase systems

In this study, the interplay of two linked equilibria is examined, one concerning an aqueous two-phase system (ATPS) composed of poly(ethylene glycol) (PEG) and salt employed to partition plasmid DNA (pDNA), and the other a potential structural transition of pDNA depending on PEG and salt concentration and other system parameters. The boundary conditions for pDNA partitioning are set by PEG and salt concentrations, PEG molecular weight, pH, and temperature. While investigating these parameters, it was found that a small increase/decrease of the respective values led to a drastic and significant change in pDNA behavior. This behavior could be attributed to a coil-globule transition of the pDNA triggered by the respective phase conditions. The combination of this structural change, aggregation effects linked to the transition process, and the electrostatic potential difference found in PEG-salt systems thus offers a sensitive way to separate nucleic acid forms on the basis of their unique property to undergo coil-globule transitions under distinct system properties.     

Extraction and determination of morphine in compound liquorice using an aqueous two-phase system of poly(ethylene glycol)/K2HPO4 coupled with HPLC

An aqueous two-phase system (ATPS) of poly(ethylene glycol) (PEG)/K₂HPO₄ coupled with high performance liquid chromatography (HPLC) method was developed for the separation and determination of morphine in compound liquorice. Morphine and its analogs were used as model compounds to investigate influence of various factors on extraction behaviors of ATPS, such as the types and concentrations of salts, PEG molecular mass, temperature and pH. It was observed that the types of salt had much influence on extraction efficiencies of morphine and its analogs. The results indicated that hydrophobic force cooperating with hydrogen bond interaction between analytes and phases played important role in extraction process. In the optimal system of containing 0.5 g PEG2000 and 1.5 g K₂HPO₄, the recoveries of the spiked standards for the analytes were all 91.7-100.3% with relative standard deviation of 1.0-3.0%. Morphine in compound liquorice was determined by the proposed method and the results were consistent with those of LC-MS method. Compared with conventional liquid-liquid extraction or solid-phase extraction, this extraction method can be completed in one operation and is low-cost. Since the entire extraction process is organic solvent-free, this new technique is environmental friendly.     

Synthesis, DNA intercalation and europium(III)-triggered DNA photocleavage by a bis-proflavine succinamide conjugate

We describe the synthesis of a bis-proflavine derivative containing a succinamide linking chain. Levels of pUC19 plasmid DNA photocleavage by this compound are significantly enhanced in the presence of Eu³⁺ (350 nm, 22 °C, pH 7.0). UV-visible spectrophotometric studies of the ligand with calf thymus DNA show bathochromic-shifts and hypochromicity in the major absorption bands of the bis-proflavine derivative. Viscosimetric analysis of the helical extension of sonicated calf thymus DNA agrees with a monofunctional intercalation process.     

A sol-gel immobilization of nano and micron size sorbents in poly(dimethylsiloxane) (PDMS) microchannels for microscale solid phase extraction (SPE)

Sorbent particles consisting of nano and micro silica, and micron size octadecylsilica (ODS) were immobilized using sol-gel chemistry onto poly(dimethylsiloxane) (PDMS) microfluidic channels to serve as μ-chip solid phase extraction (SPE) devices. Extraction, preconcentration and purification of biological and chemical analytes were carried out using these. Micro and nano scale silica-immobilized μ-SPE were used for the extraction/purification of DNA from recombinant Escherichia coli crude lysate. The average DNA recovery was 77 ± 9% (X ± R.S.D.) for the micron size silica particles and 70 ± 5% (X ± R.S.D.) for the nano silica particles. The extracted DNA could be amplified by polymerase chain reaction (PCR) whereas the DNA from the crude lysate solution could not be. This was a testimony to the purification capability of the μ-SPE device. ODS immobilized μ-SPE were used to study the extraction efficiency (EE) and enhancement factor (EF) for three groups of organic compounds, aromatics, phenols and carboxylic acids. They showed poor recovery and low enrichment because the analytes sorbed into the PDMS and were not quantitatively extracted.     

Application of a new hybrid organic-inorganic monolithic column for efficient deoxyribonucleic acid purification

A new hybrid organic-inorganic monolithic column for efficient deoxyribonucleic acid (DNA) extraction was prepared in situ by polymerization of N-(β-aminoethyl)-γ-aminopropyltriethoxysilane (AEAPTES) and tetraethoxysilane (TEOS). The main extraction mechanism was based on the Coulombic force between DNA and the amino silica hybrid monolithic column. DNA extraction conditions, such as pH, ion concentration and type, and loading capacity, were optimized online by capillary electrophoresis with laser-induced fluorescence detection. Under optimal condition, a 6.0-cm monolithic column provided a capacity of 48 ng DNA with an extraction efficiency of 74 ± 6.3% (X ± RSD). The DNA extraction process on this monolithic column was carried out in a totally aqueous system for the successful purification of DNA and removal of proteins. The PBE2 plasmid could be extracted from Bacillus subtilis (B. subtilis) crude lysate within 25 min, and the purified DNA was suitable for the amplification of a target fragment by polymerase chain reaction. This study demonstrates a new attractive solid-phase support for DNA extraction to meet the increasingly miniaturized and automated trends of genetic analyses.     

A green deep eutectic solvent-based aqueous two-phase system for protein extracting

As a new type of green solvent, deep eutectic solvent (DES) has been applied for the extraction of proteins with an aqueous two-phase system (ATPS) in this work. Four kinds of choline chloride (ChCl)-based DESs were synthesized to extract bovine serum albumin (BSA), and ChCl-glycerol was selected as the suitable extraction solvent. Single factor experiments have been done to investigate the effects of the extraction process, including the amount of DES, the concentration of salt, the mass of protein, the shaking time, the temperature and PH value. Experimental results show 98.16% of the BSA could be extracted into the DES-rich phase in a single-step extraction under the optimized conditions. A high extraction efficiency of 94.36% was achieved, while the conditions were applied to the extraction of trypsin (Try). Precision, repeatability and stability experiments were studied and the relative standard deviations (RSD) of the extraction efficiency were 0.4246% (n = 3), 1.6057% (n = 3) and 1.6132% (n = 3), respectively. Conformation of BSA was not changed during the extraction process according to the investigation of UV–vis spectra, FT-IR spectra and CD spectra of BSA. The conductivity, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to explore the mechanism of the extraction. It turned out that the formation of DES–protein aggregates play a significant role in the separation process. All the results suggest that ChCl-based DES-ATPS are supposed to have the potential to provide new possibilities in the separation of proteins.     

Evaluation of a PEG/hydroxypropyl starch aqueous two‐phase system for the separation of monoclonal antibodies from cell culture supernatant

In this study, an aqueous two‐phase system (ATPS) with PEG and hydroxypropyl starch (HPS) was used to separate monoclonal antibody (mAb) from Chinese hamster ovary cell culture supernatant. The phase diagram of the PEG/HPS ATPS was determined, and the effects of NaCl addition were investigated. The results showed that NaCl addition could lead to a shift of the binodal curve and that phase separation would occur at higher PEG and HPS concentrations. The effects of NaCl addition, pH, and the load of cell supernatant on the partitioning of mAb in a PEG/HPS ATPS were investigated. It was found that with 6% cell supernatant and 15% NaCl addition at pH 6.0, the yield of mAb in the upper phase was 96.7% with a purity of 96.0%. The back‐extraction of mAb with a PEG/phosphate ATPS were also studied, and the results showed that after the two‐step extraction with ATPSs the purity of mAb could reach 97.6 ± 0.5% with a yield of 86.8 ± 1.0%, which was comparable to the purification with Protein A chromatography. These results indicate that the two‐step extraction with PEG/HPS and PEG/phosphate ATPSs might be a promising alternative for the separation of mAb from cell culture supernatant.     

Affinity partitioning of human antibodies in aqueous two-phase systems

The partitioning of human immunoglobulin (IgG) in a polymer-polymer and polymer-salt aqueous two-phase system (ATPS) in the presence of several functionalised polyethylene glycols (PEGs) was studied. As a first approach, the partition studies were performed with pure IgG using systems in which the target protein remained in the bottom phase when the non-functionalised systems were tested. The effect of increasing functionalised PEG concentration and the type of ligand were studied. Afterwards, selectivity studies were performed with the most successful ligands first by using systems containing pure proteins and an artificial mixture of proteins and, subsequently, with systems containing a Chinese hamster ovary (CHO) cells supernatant. The PEG/phosphate ATPS was not suitable for the affinity partitioning of IgG. In the PEG/dextran ATPS, the diglutaric acid functionalised PEGs (PEG-COOH) displayed great affinity to IgG, and all IgG could be recovered in the top phase when 20% (w/w) of PEG 150-COOH and 40% (w/w) PEG 3350-COOH were used. The selectivity of these functionalised PEGs was evaluated using an artificial mixture of proteins, and PEG 3350-COOH did not show affinity to IgG in the presence of typical serum proteins such as human serum albumin and myoglobin, while in systems with PEG 150-COOH, IgG could be recovered with a yield of 91%. The best purification of IgG from the CHO cells supernatant was then achieved in a PEG/dextran ATPS in the presence of PEG 150-COOH with a recovery yield of 93%, a purification factor of 1.9 and a selectivity to IgG of 11. When this functionalised PEG was added to the ATPS, a 60-fold increase in selectivity was observed when compared to the non-functionalised systems.     

Separation and purification of aloe polysaccharides by a combination of membrane ultrafiltration and aqueous two-phase extraction

A two-step process was developed for the purification of polysaccharides from the pulp of Aloe varavia using aqueous two-phase system (ATPS) extraction and a novel copolymer ultrafiltration membrane. The first step was ATPS under optimal separations conditions using a total composition of 18% PEG2000, 25% ammonium sulfate, pH 3.0, and 0.3 M NaCl. To form the copolymer membrane, poly(acrylonitrile-acrylamide-styrene) was prepared by solution polycondensation using azoisobutyronitrile as initiator. Then, membranes were formed from the dissolved copolymer by the phase inversion method. Copolymer structure was investigated by infrared spectrum and thermogravimetric analysis (TGA). The copolymer membrane surface and cross section were observed by scanning electron microscopy. The water flux of this membrane was 26.33 mL/(cm² h), and retention was 96% for bovine serum albumin and 34% for dextran T40000. The separation and purification of aloe polysaccharide were carried using this copolymer membrane following ATPS. The TGA of aloe polysaccharide demonstrated a high purity of the polysaccharide. By gas chromatographic analysis, it was shown that mannose is the main monosaccharide in the aloe polysaccharide, and only a few glucose residues are present.     

Simultaneous determination of polysorbate 20 and unbound polyethylene-glycol in protein solutions using new core–shell reversed phase column and condensation nucleation light scattering detection

A novel fast and sensitive method has been developed for the specific simultaneous determination of polysorbate 20 (Tween 20) and unbound polyethylene-glycol (PEG) from liquid formulations in the presence of proteins and excipients. The quantitative determination is based on a fast liquid chromatographic (HPLC) separation and condensation nucleation light scattering detection (CNLSD or NQAD™). The method uses a Kinetex core–shell column (100 mm × 3 mm, 2.6 μm) and methanol–water–trifluoroacetic acid mobile phase. The rapid HPLC-CNLSD method presented here is suitable for quantifying polysorbate 20 in the range of 10–60 μg/ml and unbound PEG in the range of 2–40 μg/ml in protein solutions within good manufacturing practices (GMP) of the pharmaceutical industry.     

Dispersive liquid–liquid–liquid microextraction combined with liquid chromatography for the determination of chlorophenoxy acid herbicides in aqueous samples

A novel sample preparation method “Dispersive liquid–liquid–liquid microextraction” (DLLLME) was developed in this study. DLLLME was combined with liquid chromatography system to determine chlorophenoxy acid herbicide in aqueous samples. DLLLME is a rapid and environmentally friendly sample pretreatment method. In this study, 25 μL of 1,1,2,2-tetrachloroethane was added to the sample solution and the targeted analytes were extracted from the donor phase by manually shaking for 90 s. The organic phase was separated from the donor phase by centrifugation and was transferred into an insert. Acceptor phase was added to this insert. The analytes were then back-extracted into the acceptor phase by mixing the organic and acceptor phases by pumping those two solutions with a syringe plunger. After centrifugation, the organic phase was settled and removed with a microsyringe. The acceptor phase was injected into the UPLC system by auto sampler. Fine droplets were formed by shaking and pumping with the syringe plunger in DLLLME. The large interfacial area provided good extraction efficiency and shortened the extraction time needed. Conventional LLLME requires an extraction time of 40–60 min; an extraction time of approximately 2 min is sufficient with DLLLME. The DLLLME technique shows good linearity (r² ≥ 0.999), good repeatability (RSD: 4.0–12.2% for tap water; 5.7–8.5% for river water) and high sensitivity (LODs: 0.10–0.60 μg/L for tap water; 0.11–0.95 μg/L for river water).     

Influence of magnetic field on aqueous two-phase extraction of horse ferritin in the polyethylene glycol/hydroxyethyl starch system

The presented experiments show the model of expectation of equine spleen ferritin extraction in a new aqueous two-phase system which was formed by mixing polyethylene glycol (PEG) and hydroxyethyl starch (HES). The tendency of the protein to migrate in the analyzed systems was dependent on the concentrations of HES and PEG as well as PEG molecular weight. The highest concentration of ferritin in the top phase (rich in PEG) was recorded in the system composed of 6% PEG 3000 and 3% HES. The obtained concentration was 0.88 mg mL⁻¹. The lowest concentration was 0.42 mg mL⁻¹ in the system composed of 5% PEG 6000 and 1% HES.     

Solvatochromic relationship: Prediction of distribution of ionic solutes in aqueous two-phase systems

Partition ratios of several ionic compounds in 20 different polymer/polymer aqueous two-phase systems (ATPS) containing 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.4, were determined. The differences between the electrostatic properties of the phases in all the ATPS were estimated from partitioning of the homologous series of dinitrophenylated-amino acids. Also the solvatochromic solvent parameters characterizing the solvent dipolarity/polarizability (π*), solvent hydrogen-bond donor acidity (α), and solvent hydrogen-bond acceptor basicity (β) of aqueous media were measured in the coexisting phases of the ATPS. The solute-specific coefficients for the compounds examined were determined by the multiple linear regression analysis using the modified linear solvation energy relationship equation. The minimal number of ATPS necessary for determination of the coefficients was established and 10 ATPS were selected as a reference ATPS set. The solute-specific coefficients values obtained with this reference set of ATPS were used to predict the partition ratios for the compounds in 10 ATPS not included in the reference set. The predicted partition ratios values were compared to those determined experimentally and found to be in good agreement. It is concluded that the presented model of solute–solvent interactions as the driving force for solute partitioning in polymer/polymer ATPS describes experimental observations with 90–95% accuracy.     

Protein separation using toroidal columns by type-J synchronous counter-current chromatography towards preparative separation

Separation of large bioactive molecules such as proteins, DNAs and RNAs using aqueous two-phase systems (ATPSs) and liquid–liquid partition-based counter-current chromatography (CCC) can avoid risks of sample loss and denaturation, and greatly reduce processing time. We have constructed toroidal columns (length 26–140 m, column volume 51–280 ml, bore size 1.6 mm) suitable for mounting onto a commercially available preparative CCC apparatus. With the use of an ATPS containing 12.5% (w/w) PEG1000 and 12.5% (w/w) K₂HPO₄ and at a rotational speed of 800 rpm for the rotor of the CCC device, the lower phase (i.e. the phosphate-enriched phase) has been used as the mobile phase and a pair of proteins, myoglobin and lysozyme, as model proteins for demonstrating the separation capability of the CCC system. For a toroidal column with a length of 53.5m and a column volume of 107.5 ml, and operated for the Coriolis force parallel flow mode at 0.62 ml/min, protein sample loading (containing 2.2 mg/ml myoglobin and lysozyme, respectively) at 1.7% and 7.4% to the column volume led to peak resolution (with theoretical plate number TP and stationary phase retention S_f shown in the parenthesis) of R_s = 1.5 (N = 211 and N = 113 TP for myoglobin and lysozyme, respectively, and S_f = 45.0%), and R_s = 1.4 (218 and 152 TP, and S_f = 34.0%). However, further increase of the loading to 13% failed to separate the two proteins. Although proteins eluted at positions predictable from the distribution coefficients, they showed broader peaks when compared with small dipeptides under identical CCC operating conditions. This confirms that the molecular weight of the partitioned species is an important factor causing peak broadening on CCC chromatograms. These results paved the way for further scaling-up toroidal CCC columns for processing larger quantities of samples containing large biomolecules.     

Hollow fiber supported ionic liquid membrane microextraction for determination of sulfonamides in environmental water samples by high-performance liquid chromatography

By using ionic liquid as membrane liquid and tri-n-octylphosphine oxide (TOPO) as additive, hollow fiber supported liquid phase microextraction (HF-LPME) was developed for the determination of five sulfonamides in environmental water samples by high-performance liquid chromatography with ultraviolet detection The extraction solvent and the parameters affecting the extraction enrichment factor such as the type and amount of carrier, pH and volume ratio of donor phase and acceptor phase, extraction time, salt-out effect and matrix effect were optimized. Under the optimal extraction conditions (organic liquid membrane phase: [C₈MIM][PF₆] with 14% TOPO (w/v); donor phase: 4 mL, pH 4.5 KH₂PO₄ with 2 M Na₂SO₄; acceptor phase: 25 μL, pH 13 NaOH; extraction time: 8 h), low detection limits (0.1–0.4 μg/L, RSD ≤ 5%) and good linear range (1–2000 ng/mL, R² ≥ 0.999) were obtained for all the analytes. The presence of humic acid (0–25 mg/L dissolved organic carbon) and bovine serum albumin (0–100 μg/mL) had no significant effect on the extraction efficiency. Good spike recoveries over the range of 82.2–103.2% were obtained when applying the proposed method on five real environmental water samples. These results indicated that this present method was very sensitive and reliable with good repeatabilities and excellent clean-up in water samples. The proposed method confirmed hollow fiber supported ionic liquid membrane based LPME to be robust to monitoring trace levels of sulfadiazine, sulfamerazine, sulfamethazine, sulfadimethoxine and sulfamethoxazole in aqueous samples.     

Analysis of capsaicin and dihydrocapsaicin in peppers and pepper sauces by solid phase microextraction–gas chromatography–mass spectrometry

A simple method for the analysis of capsaicin and dihydrocapsaicin in peppers and pepper sauces by solid phase microextraction–gas chromatography–mass spectrometry has been developed. A novel device was designed for direct extraction solid phase microextraction in order to avoid damage to the fiber. The analysis was performed without derivatization for the gas chromatography–mass spectrometry analysis. Selection fiber, extraction temperature, extraction time and pH, were optimized. The method was linear in the range 0.109–1.323 μg/mL for capsaicin and 0.107–1.713 μg/mL for dihydrocapsaicin with correlation coefficient up to r = 0.9970 for both capsaicinoids. The precision of the method was less than 10%. The method was applied to the analysis of 11 varieties of peppers and four pepper sauces. A broad range of capsaicin (55.0–25 459 μg/g) and dihydrocapsaicin (93–1 130 μg/g) was found in the pepper and pepper sauces samples (4.3–717.3 and 1.0–134.8 μg/g), respectively.