Plasma normal phase HPLC

Miyazawa et al,143 determined the Amadori compounds from dioleylphos-phatidylethanolamine by normal-phase HPLC as the 3-methyl-2-benzothiazolinone hydrazones (MBTH) through their UV absorption (318 nm) with detection limits of 4.5 and 5.3 ng for the fructosyl (F) and the lactulosyl (L) compound, respectively, and linear ranges of up to about 2 ng. Using their method, they obtained the following results infant formula, F 32-112, L 49-88 ng g mayonnaise, F 12.2 ng g-1 chocolate, F 3.9, L 1.5 ng g-1 cow s milk, L 0.079 /xg mL-1 soybean milk, F 0.24, L 0.13 /tg mL-1 and rat plasma, F 0.23 ng mL-1. Significant amounts were not detected in human milk. 

The normal phase HPLC (20% chloroform in heptane) could separate A9-tetrahydrocannabinol from monohydrox-ylated metabolites and from 11-hydroxy-A tetrahydrocannabinol. However, a minor overlap could be avoided by collecting the tetrahydrocannabinol 1 in a slightly narrower volume range. The prior heptane extraction of alkalinized plasma had separated these non-polar constituents from any acidic metabolite. This separation of plasma extracts and normal phase HPLC collection of volumes in the appropriate range resulted in a substantial reduction in GLC background from plasma components for derivatized tetrahydrocannabinol analyzed with electron capture (63 1) detection. 

Plasma samples obtained from dogs, administered A9-tetrahydrocannabinol solutions intraveneously, were analyzed by the electron capture GLC in accordance with the modified procedures described herein that included extraction, normal phase HPLC separation and derivati-zation except that no internal standard was added. 

The recovery of 1 from the heptane extract of dog plasma by normal phase HPLC was reproducible over the range of plasma concentrations studied. Equivalent overall recoveries were obtained by both radiochemical analysis (83.7 1.8% SE) and electron-capture GLC analysis (84.0 4.9% SE) of the derivatized tetrahydrocannabinol. ... 

The plasma of a dog intravenously administered solutions of l c-A -tetrahydrocannabinol was monitored with time after heptane extraction by both radiochemical analysis and electron-capture GLC of the derivative of the appropriately collected eluate fraction from normal phase HPLC. Typical plots of the time course of the results from both methods are given in Figure 7. 

Figure 7. Semilogarithmic plots of fraction of the b -THC, 2.0 mg/kg dose/mL of plasma, vs. time for Dog A from (O), the liquid scintillation analysis of the total I4C collected as ts -THC on normal-phase HPLC and from (9), the electron-capture GLC of the derivatized HPLC collected fraction. The values were corrected for the fractions of extracts and total collection range used (15).

Both normal phase and reverse phase HPLC methods were studied as analytical techniques for analysis of I and VI in human plasma. Normal phase was found to be more satisfactory for separation of I from plasma constituents whereas reverse phase was the choice for VI. Although reverse phase HPLC could be used to simultaneously assay for both I and VI when placed directly on the instrument, it was not practical for analysis of plasma extracts. 

Retinoic acid, an endogenous retinoid, is a potent inducer of cellular differentiation. Because cancer is fundamentally a loss of cellular differentiation, circulating levels of retinoic acid could play an important role in chemoprevention. However, physiological concentrations are typically below the limits of HPLC detection. Sensitive techniques, such as negative chemical ionization (NCI) GC/MS have been employed for quantification, but cause isomerization and also fail to resolve the cis and trans isomers of retinoic acid. Normal phase HPLC can resolve the cis and trans isomers of retinoic acid without isomerization, and mobile phase volatility makes it readily compatible with the mass spectrometer. Based on these considerations, a method combining microbore normal phase HPLC separation with NCI-MS detection was developed to quantify endogenous 13-cis and all-trans retinoic acid in human plasma. The limit of detection was 0.5 ng/ml, injecting only 8 pg of retinoic acid onto the column. The concentration of 13-cis retinoic acid in normal, fasted, human plasma (n=13) was 1.6 +/- 0.40 ng/ml. 

Most assays for the quantification of endogenous levels of 13-cis and all trans retinoic acid utilize GC/MS. This technique is highly sensitive, but GC isomer resolution is an inherent problem (22). Therefore, unequivocal quantification of cis and trans retinoic acid levels is impossible. The use of HPLC can eliminate isomerization, but lacks sensitivity. Therefore, HPLC in combination with MS should provide a highly sensitive method of quantification without isomerization. In this report, we describe the use of microbore normal phase HPLC/NCI-MS to quantify endogenous levels of 13-cis and all trans retinoic acid in human plasma. 

Several investigators have developed assays for the quantification of all trans retinoic acid in human blood, plasma or serum. Nelson et al. (35) developed a colorimetric assay, but could not detect retinoic acid under physiological conditions. DeRuyter et al. (36) found 1 to 3 ng/ml of all trans retinoic acid in fasted human serum using normal phase HPLC. Chiang (37) validated an assay for all trans retinoic acid in plasma, but could not detect any under normal physiological conditions using 10 ml of plasma. 

Meyer, E. Lambert, W.E. De Leenheer, A.P. Simultaneous determination of endogenous retinoic acid isomers and retinol in human plasma by isocratic normal-phase HPLC with ultraviolet detection. Clin.Chem., 1994, 40, 48—51... 

Kwon et al. (1996) used normal-phase HPLC to separate and quantify sphingomyelin (SPH). Flow injection ES-MS was then used to determine the SPH molecular species composition of isolated rat pancreatic islets. Kwon et al. concluded that sphingomyelin hydrolysis is not involved in the signalling pathway whereby cytokine interleukin-1 induces the overproduction of nitric oxide by pancreatic islets. They reported four molecular species of SPH SPH (16 0/d-18 l), (18 0/d-18 l), (22 0/d-18 l) and (24 0/d-18 l). They assumed that sphingosine (18 1) is the LCB however, studies of SPH from bovine brain (Jungalwala, Evans and McCluer, 1984), bovine milk (Morrison, 1969) and human plasma (Sweeley, 1963), show that there exist several LCB species, saturated as well as unsaturated, with one or two hydroxyl... 

Fig. 3 Resolution of RA isomers by normal- and reverse-phase HPLC (A) A normal-phase HPLC column (Econosphere 3 mm, 0.45 x 15 cm) was eluted with hexane/ methylene chloride/acetic acid (95/5/0 2 v/v) at 2 mL/mm (B) A reverse-phase HPLC column (Suplex pkb-100,5 mm, 0 46 x 25 cm) was eluted with acetonitnle/methanol/ water/chloroform/acetic acid (17/68/10/5/0 5 v/v) at 2 mL/min RAs were detected at 340 nm The example depicts a typical analysis of 2 mL of 2-d-old calf plasma (8).

Meyer et al. analyzed plasma retinol plus endogenous aW-trans and i-cis retinoic acid isocratically by normal-phase HPLC, using hexane 2-propanol acetic acid as mobile phase (90). Extraction of the retinoic acids required acidification of the sample however, too much acid can result in dehydration of retinol to anhydroretinol, and hydrolysis of endogenous retinoyl P-glucuronide (90). A synthetic retinoid sulfonic acid was used as internal standard. By using absorbance at 350 nm, limits of detection were 1.7 nM (0.5 (ig/L) for retinoic acid isomers, 35 nM (10 Xg/L) for retinol. 13-Demethyl retinoic acid has also been used as internal standard (142). Lanvers et al. used normal-phase HPLC with gradient elution (hexane 2-propanol glacial acetic acid) to analyze 13-c/x retinoic acid, 9-cis retinoic acid, aW-trans retinoic acid, retinol, and the 4-oxo metabolites of the retinoic acid isomers (143). Plasma samples were treated with ethanol to denature proteins, and then were extracted with hexane after addition of saturated ammonium sulfate solution (pH 5). 

MA BeUsaiio, G Azar, G Oriani, GP Pizzuti, L Sacchetti. Simultaneous evaluation of vitamins A and E in human plasma hy normal phase HPLC. BoU Soc Ital Biol Sper 69 641-647, 1993. 

Numerous high pressure Hquid chromatographic techniques have been reported for specific sample forms vegetable oHs (55,56), animal feeds (57,58), seta (59,60), plasma (61,62), foods (63,64), and tissues (63). Some of the methods requite a saponification step to remove fats, to release tocopherols from ceHs, and/or to free tocopherols from their esters. AH requite an extraction step to remove the tocopherols from the sample matrix. The methods include both normal and reverse-phase hplc with either uv absorbance or fluorescence detection. AppHcation of supercritical fluid (qv) chromatography has been reported for analysis of tocopherols in marine oHs (65). 

Plasma. Albendazole (ABZ), albendazole-sulfone (ABZ-S02), albendazole 2-aminosulfone (ABZ-NH2, marker metabolite) and albendazole-sulfoxide (ABZ-SO), were extracted from the plasma using a perchloric acid precipitation method followed by a solid phase extraction procedure. The residue levels were quantitated using a normal or reversed phase HPLC method with fluorescence detection. 

Vitamin K HPLC has provided the first assay of the phylloquinones and menaquinones that constitute vitamin K in plasma. Phylloquinone circulates bound to lipoproteins from which it can be extracted with hexane after ethanol protein precipitation. Removal of co-eluted lipids can be achieved with normal-phase cartridge columns. Reversed-phase HPLC is almost universally used for vitamin K measurement. Either UV (270 nm) or electrochemical detection is suitable. Electrochemical detection uses the reductive mode ( —1.3 V) to convert the quinone moiety to hydroquinone the main disadvantage being the need to remove oxygen from the mobile phase. 

Matrix effect is a phrase normally used to describe the effect of some portion of a sample matrix that causes erroneous assay results if care is not taken to avoid the problem or correct for it by some mechanism. The most common matrix effects are those that result in ion suppression and subsequent false negative results. Ion enhancement may lead to false positive results.126 127 Several reports about matrix effects include suggestions on what can cause them and how to avoid them.126-147 While various ways to detect matrix effects have been reported, Matuszewski et al.140 described a clear way to measure the matrix effect (ME) for an analyte, recovery (RE) from the extraction procedure, and overall process efficiency (PE) of a procedure. Their method is to prepare three sets of samples and assay them using the planned HPLC/MS/MS method. The first set is the neat solution standards diluted into the mobile phase before injection to obtain the A results. The second set is the analyte spiked into the blank plasma extract (after extraction) to obtain the B results. The third set is the analyte spiked into the blank plasma before the extraction step (C results) these samples are extracted and assayed along with the two other sets. The three data sets allow for the following calculations ... 

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