Analysis by precolumn derivatization and HPLC separation of melanotropin potentiating factor using FMOC-C1

has provided insights into the operation and modulation of both transcellular and paracellular routes and the utilization of both carrier-mediated transmembrane pathways and endocytic mechanisms. While there is evidence that small amounts of intact protein can cross intestinal enterocytes via endocytic vesicles, it is unlikely that in the adult human gut this process occurs by a direct transcytotic pathway operating from the mucosal to the serosal membrane, in contrast to the pathway in suckling animals. Current evidence on the receptor-mediated endocytosis of internalizing intrinsic factor-cobalamin from the mucosal membrane, with the ultimate transport of cobalamin into the blood suggests that the intracellular destination of cobalamin-drug conjugates will be the lysosome, and that such conjugates will need to survive lysosomal degradation, penetrate the lysosomal membrane and be exocytosed from the cell. The transcytosis of intact proteins, particles and pathogens through intestinal M-cells has been well documented and increased transport of proteins by absorptive endocytosis demonstrated. The transport of proteins to the vicinity of phagocytic cells, by this pathway, suggests that it will have more relevance to the delivery of vaccines than the delivery of therapeutic proteins to the systemic circulation. Recent evidence on the utilization of amino acid and peptide carriers, the interaction of lipid carriers with intestinal cells, and the exploitation of transient changes in tight-junctional permeability will also be reviewed. The potential for utilizing transepithelial transport routes to safely achieve required levels of selected peptidergic drugs in the blood or lymph, will be discussed.

ANALYSIS BY PRECOLUMN DERIVATIZATION AND HPLC SEPARATION OF MELANOTROPIN POTENTIATING FACTOR USING FMOC-Cl J.R.E. Lewis, J.S. Morley and R.F. Venn Pain Relief Foundation, Rice Lane, Liverpool L9 7A E (U.K.)

Melanotropin potentiating factor (MPF) is the C-terminal tetrapeptide (Lys-Lys-Gly-Glu) of human /3-endorphin. It is a putative neurotrophic factor and has been implicated in the initiation of urodele limb regeneration [ 11. Any assay will have to measure the peptide at the subpicomole level and differentiate it from a large number of similar and precursor peptides in a physiological environment. The problems of cross reactivity in a radioimmunoassay, specifically with /?-endorphin and lipotropin fragments would be considerable. MPF itself is non-immunogenic and may partially disable the immune system, making antisera difficult to raise. It was therefore decided to use precolumn derivatization and HPLC separation employing 9-fluorenylmethylchloroformate (FMOCCl), a highly fluorescent base labile reagent that reacts with the three free amino groups rapidly and in high yield under mild conditions. The assay is now being used to determine MPF concentrations down to a level of a few hundred femtomoles from cell culture media and CNS tissues from animal and human origin. Problems encountered in the form of interfering peaks have been overcome by exploiting the unusual physical properties of the peptide (small size and highly charged nature) and carrying out a prederivatization sample clean up step using ion-exchange or size-exclusion chromatography. FMOC-MPF fluorescence is linear with concentration up to at least 10 picomoles and leads to an ultimate sensitivity of 100 femtomoles at a signal to noise ratio of 3 :1. This approach fulfils the above criteria for the assay, negates many of the problems of specificity, and has the added advantage of being easily adapted to measure related peptides and artificial an-

alogues of MPF simultaneously. Reference: 1 J.S. Morley and D.M. Ensor, Neurotrophic effects of /?-endorphin C-terminal tetrapeptide, Neuropeptides, 8 (1989) 45-49.

SAMPLE

PRETREATMENT

D.S. Stegehuis,

U.R. Tjaden

Center for Bio-Pharmaceutical

IN PEPTIDE

BIOANALYSIS

and J. van der Greef

Sciences, Division of Analytical Chemistry, P. 0. Box 9502,

2300 RA Leiden (The Netherlands)

Endorphins are endogenous peptides working at extremely low levels in the central nervous system. Due to their high potency and low side effects they are of growing importance. For study of the working mechanism, determination of endogenous levels is necessary. The dodecapeptide desenkephalin-y-endorphin (DEyE) is chosen as test compound, since this peptide combines a wide variety of problems encountered in the bioanalysis of peptides. This peptide is unstable in biological matrices, its half-life in plasma being only a few minutes, demonstrating the degradation activity. Therefore the first priority is elimination of degrading factors, which can be achieved by inhibiting the enzyme activity, for instance by adding perchloric acid to the sample, immediately after collection. The sample has to be cleared from all high molecular compounds that can disturb the HPLC system by blocking the column. The excess of amino function containing compounds has also to be removed, since such compounds can interfere with the detection after post-column derivatization with o-phthaldialdehyde (OPA). Continuous-flow dialysis and on-line gel permeation have been investigated for elimination of high molecular compounds. The latter technique appeared to be superior with respect to selectivity and recovery. The analytic component containing fraction is concentrated by solid phase isolation. After gradient elution, heart cutting of the compounds of interest before isocratic elution results in limits of determination, obtained with conventional fluorescence detection, in the nanogram/ml range. This level can be lowered to the subnanogram range by the application of laser induced fluorescence detection (LIF ). The method is still under investigation.

LASER INDUCED C.M.B.

FLUORESCENCE

van den Beld, U.R. Tjaden

Center for Bio-Pharmaceutical

REACTION

DETECTION

IN BIOANALYSIS

and J. van der Greef

Sciences, Division of Analytical Chemistry, P. 0. Box 9502,

2300 RA Leiden (The Netherlands)

Nowadays laser induced fluorescence (LIF) detection is successfully applied in combination with several separation methods. In conventional HPLC systems, detection can be performed with conventional quartz flow-cells. With fused silica capillary (I.D. lo-200 pm) as the column, as for instance in high performance capillary electrophoresis (HPCE), on-column detection yields probe volumes in the nanoliter or even picoliter range. In this study a continuous-wave argon-ion laser