Reversed-phase HPLC with postcolumn

Thiamine (vitamin Bj) occurs in foods in free and bound forms, the free form predominates in cereals and plants, whereas the pyrophosphate ester is the main form in animal products. Acid hydrolysis is required to release thiamine from the food matrix. Enzymatic hydrolysis is then needed to convert phosphate esters to thiamine. Prior to CE analysis it is necessary to clean up samples by using ethanol to precipitate protein and by passing through an ion-exchange resin. Thiamine has been determined in meat and milk samples using MEKC with ultraviolet (UV) detection at 254 nm, obtaining comparable sensitivity to that achieved by HPLC using an ion-pair reversed-phase column with postcolumn derivat-ization and fluorescence detection. 

A reversed-phase HPLC Cl8 or C30 narrow-bore column is typically used for LC/MS with APCI. Details about chromatography columns used for carotenoids are contained in unit F2.3. For most APCI systems, the optimum flow rate into a mass spectrometer or tandem mass spectrometer equipped with APCI, as controlled by a syringe pump or HPLC pump, is usually between 100 and 300 pl/min, which is ideal for narrow-bore HPLC columns. Larger diameter columns should be used with a flow splitter postcolumn to reduce the solvent flow into the mass spectrometer. For example, if a 4.6 mm i.d. column was used at a flow rate of 1.0 ml/min, then the flow must be split postcolumn 5 1 so that only 200 pl/min enters the mass spectrometer. 

All of the fat-soluble vitamins, including provitamin carotenoids, exhibit some form of electrochemical activity. Both amperometry and coulometry have been applied to electrochemical detection. In amperometric detectors, only a small proportion (usually <20%) of the electroactive solute is reduced or oxidized at the surface of a glassy carbon or similar nonporous electrode in coulometric detectors, the solute is completely reduced or oxidized within the pores of a graphite electrode. The operation of an electrochemical detector requires a semiaqueous or alcoholic mobile phase to support the electrolyte needed to conduct a current. This restricts its use to reverse-phase HPLC (but not NARP) unless the electrolyte is added postcolumn. Electrochemical detection is incompatible with NARP chromatography, because the mobile phase is insufficiently polar to dissolve the electrolyte. A stringent requirement for electrochemical detection is that the solvent delivery system be virtually pulse-free. 

Most amino acids react with ninhydrin at ambient temperatures to form a blue color that becomes purple on heating. However, proline and hydroxyproline yield yellow compounds that are measured at a different wavelength. Other postcolumn derivatizations use fluorogenic reagents, such as o-phthaldialdehyde or fluorescamine. Precolumn derivatization techniques using o-phthaldialdehyde, dansyl, phenyl isothiocyanate, or 9-fluorenylmethyl chloroformate derivatives have been used with reversed-phase HPLC. Electrochemical detection has also been coupled with derivatization methods to enhance analytical sensitivity. 

More recently, the use of HPLC with postcolumn reaction with ABTS has successfully determined the peracids up to Ci2- A Merck LiChroSorb RP18 reversed-phase column (125 X 4 mm, 5 /rm particle size) was used with acetonitrile-water gradient elution (25 to 100 to 25 %) at a flow rate of 1.4 ml min. To optimize peak shape, 2% acetic acid and 1 % tetrahydrofuran were added to the water eluent. A turbomixing chamber was used to mix the eluent with the ABTS reagent for postcolumn reaction. The resulting oxidation product (green radical cation) was... 

Fluorimetric reaction detectors were developed by Reh and Schwedt and by Verbeke and Vanhee [205, 206], and fluorimetric detection has been the rule in postcolumn derivatization reactions of steroids, including the use of such fluorophores as glycinamide [207], benz-amidine [208] and 3-chloroformyl-7-methoxycoumarin [209]. Dansylated steroids were separated by reversed phase HPLC and the pre-column derivatization was complemented by a post-column treatment with peroxy-oxalate and detection by chemiluminescence [210]. 

To achieve increased sensitivity and reduced analysis time it is necessary to produce derivatives of the amino acids which can be separated by reversed-phase HPLC and then detected using fluorescent, spectrophotometric, or electrochemical detection. Both precolumn and postcolumn derivatization methods have been described with a range of derivat-izing agents including ... 

A selective and sensitive method for the determination of aliphatic anionic surfactants is reversed phase HPLC combined with postcolumn derivatization and fluorescence detection [62], After HPLC separation of the surfactants on a Q column, a UV-active cationic dye is added to the eluate in order to form fluorescent ion pairs. Then CHCI3 is added to the eluent stream as the extraction solvent for the ion pairs. The two phases are conducted through a sandwich-t) e phase separator where the major part of the organic phase is separated. Finally, the amount of ion pairs extracted into CHCI3 is determined by a fluorescence detector. 

A reversed-phase HPLC system for thiamine and its phosphates, with precolumn derivatization to thiochromes, was first described by Kawasaki s group (17,27). In this system TTP elutes first, followed by TPP, TMP, and finally thiamine. Since then, several reversed-phase HPLC systems were described with either precolumn (30-32) or postcolumn derivatization (33,34). 

A reversed-phase HPLC of thiamine phosphates followed by postcolumn fluorogenic oxidation was described by Kimura et al. (33). A pBondapak Cig column was used with a mobile phase of a 0.2 M sodium phosphate-phosphoric acid buffer (pH 4.3). The effluent was oxidized and detected fluorometrically. 

Another reversed-phase HPLC system with precolumn derivatization has been reported by Iwata and his group (31). Thiamine and its phosphates were converted to fluorophores by alkaline cyanogen bromide. The thiochromes were injected, in a neutralized solution, on an ODS silica column. The mobile phase, containing 100 mMNa2HP04-H3P04 buffer (pH 2.5) and methanol (92 8), was mixed postcolumn for alkalinization with 0.2 M NaOH-70% methanol, followed by fluorescence detection. The elution of these four thiochromes was completed in 30 min, and the detection limit was 0.1 pmol for thiamine phosphate esters and 0.05 pmol for thiamine. 

Gregory and Feldstein (94) developed an ion-paired, reversed-phase HPLC method for individual Be vitamers extracted with sulphosalicylic acid from different foods. Using a ternary solvent program, elution of nutritionally active Be vitamers from the analytical column was complete within 30 min. PLP was determined as its hydroxysulfonate derivative, following postcolumn introduction of a buffered solution of sodium bisulfite. This method was found suitable for vitamin Be analysis in foods of both plant and animal origin. Recoveries for PLP and PL from pork loin were <90% it was suggested that these vitamers were not completely released from muscle proteins, even in the presence of 5% sulfo-salicylic acid. 

Aliphatic sulfates and sulfonates may be detected with a postcolumn extraction apparatus. As the anions elute from the column, they mix with a stream containing a fluorescent cation. The cation can only be extracted into an organic phase as an ion pair with the anionic surfactant. A fluorescence detector monitors the concentration of the cation extracted. This approach makes possible the analysis of alkylsulfates, alkanesulfonates, ester sulfonates, and ether sulfates by either normal phase or reversed-phase HPLC. Gradient analysis presents no difficulty (10,26,27). Such a system was also used with methylene blue in conjunction with a visible absorbance detector at 630 nm (9). Although methylene blue does not give the sensitivity of fluorescence detection, it is more selective for surfactants. Common fluorescent cations form extractible ion pairs with nonsurfactant anions, giving rise to interference and limiting the choices available for mobile phase modification. 

Nishiyama and Kuninori [65] described a combination method of assay for penicillamine using HPLC and postcolumn reaction with 6,6 -dithiodi(nicotinic acid). Thiols were separated by HPLC on a reversed-phase column (25 cm x 4.6 mm) packed with Fine Sil 08-10, with 33 mM KH2PO4 (adjusted to pH 2.2 with H3PO4) or 33 mM sodium phosphate (pH 6.8) as the mobile phase. Detection was by postcolumn derivatization with 6,6 -dithiodi(nicotinic acid), and measurement of the absorbance of the released 6-mercaptonicotinic acid was made at 344 nm. The detection limit for penicillamine was 0.1 nmol. A comparison was made with a... 

In a method proposed by Booth et al. (141) for the determination of phylloquinone in various food types, extracted samples are subjected to silica solid-phase extraction followed, in the case of meat or milk samples, by further purification using reversed-phase solid-phase extraction or liquid-phase reduction extraction, respectively. The final test solution is analyzed by NARP-HPLC, and the fluorescent hydroquinone reduction products of phylloquinone and the internal standard are produced online using a postcolumn chemical reactor packed with zinc metal. 2, 3 -Dihydrophylloquinone, a synthetic analog of phylloquinone, is a suitable internal standard for the analysis of vegetable juice, whole milk, and spinach. Another synthetic analog, Ku23), is used for the analysis of bread and beef, because a contaminant in the test solution coelutes with dihydro-phylloquinone. 

Another approach would be to derivatize cyclamate prior to analysis. Cyclamate can be determined by HPLC and UV detection at 314 nm after conversion to A/,/V-dichlorocyclohexyl-amine. Derivatization can be carried out directly in the sample or after extraction and cleanup. /V,/V-Dichlorocyclohexylamine is separated on a reverse-phase column (Nucleosil Cl8 or Fine-pak SIL Cl8 T-5) with a mobile phase of methanol water, 8 2 v/v (43,46). Cyclamate can also be determined at 585 nm after postcolumn derivatization with methyl violet 2B as described by Lawrence and Charbonneau (16). 

A variety of detectors have been used for the HPLC determination of NOC in foods. These include UV, fluorescence, electrochemical, TEA, and various postcolumn denitrosation detectors. To be applicable for the low-ppb detection of these compounds in foods, an HPLC detector should meet the following criteria high sensitivity and specificity, responsive to all classes of NOC, linearity over a fair range of concentration, compatibility with both normal- and reversed-phase mobile phases, and minimal interference from changes in solvent composition, thereby making it amenable to solvent programming. As will be seen from the following discussion, none of the detectors currently available meet all these criteria. 

Aqueous samples analyzed by HPLC using a postcolumn reaction detector, formaldehyde separated on a reversed phase C-l 8 column derivatized with 3-methyl-2-benzothiazolinone hydrazone and detected at 640 nm (Igawa et al., 1989). (The method was developed for cloud and fogwater analysis.)... 

---