Sedimentation velocity analytical ultracentrifugation and SEDFIT/c(s): limits of quantitation for a monoclonal antibody system

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has emerged in the biopharmaceutical industry as a technique to detect small quantities of protein aggregates. However, the limits of detection and quantitation of these aggregates are not yet well understood. Although diverse factors (molecule, instrument, technique, and software dependent) preclude an all-encompassing measurement of these limits for the complete system, it is possible to use simulated data to determine the quantitation limits of the data analysis software aspect. The current study examines the performance of the SEDFIT/c(s) data analysis tool with simulated antibody monomer/dimer and monomer/aggregate systems. Under completely ideal conditions (zero noise, known meniscus, and shape factor homogeneity), the software limit of quantitation was 0.01% for the monomer/aggregate system and 0.03% for the less well-resolved monomer/dimer system. Under more realistic conditions (0.005 OD root mean square [RMS] noise, shape factor variability, and long solution column), the software limits of quantitation were 0.2 and 0.6% (0.002 and 0.006 OD) for the monomer/aggregate and monomer/dimer systems, respectively. Interestingly, diminished quantitation accuracy at very low levels of oligomer was not accompanied by deterioration of fit quality (as measured by root mean square deviation [RMSD] and residuals bitmap images).     

A reversed-phase high-performance liquid chromatographic method for the determination of aloesin, aloeresin A and anthraquinone in Aloe ferox

A reversed-phase HPLC method for the quantification of aloesin, aloeresin a and anthraquinone (as barbaloin) in Aloe ferox Miller and aloe-related products has been developed and validated. The method utilized a C18 column with a water-methanol gradient and UV detection at 297 nm. The method validation included linearity, accuracy, precision, limit of detection, limit of quantitation, specificity and standard solution stability. The method showed good linearity (r > 0.99 for all components) and recovery (>85% for all components). The detection and quantitation limits for barbaloin were determined to be 0.02 and 0.1 ppm at signal-to-noise ratios of approximately 3:1 and 10:1, respectively.     

宁南山区植被恢复对土壤团聚体水稳定及有机碳粒径分布的影响

土壤团聚作用和土壤有机碳固定之间密切相关.对宁南山区不同植被恢复措施和年限下土壤团聚体粒径分布及稳定性、土壤团聚体中有机碳及其组分分布进行了研究,探讨了有机碳及其组分对植被恢复的响应.结果表明,不同植被恢复措施下,土壤团聚体粒径分布表现为“V”字分布:＞5 mm和＜0.25 mm这两个粒径的团聚体含量最多,5-2 mm、1-0.25 mm团聚体的含量次之,2-1 mm粒径的团聚体含量最少.坡耕地的平均重量直径(MWD)最低,为1.4,其他植被恢复措施下土壤的平均重量直径MWD在1.9-3.1之间.不同的植被恢复措施下,0-20 cm土层和20-40 cm土层全土有机碳含量在7.4-17.7 g/kg之间、微生物碳含量分布在50.3-664.7 mg/kg之间、腐殖质碳含量在0.9-2.5g/kg之间.胡敏酸碳含量分布在0.2-0.6 g/kg,富里酸碳含量在0.6-1.9 g/kg之间.全土有机碳、微生物碳、腐殖质碳、富里酸碳均为坡耕地最低,其他植被恢复措施的有机碳、微生物碳、腐殖质碳、富里酸碳含量分别是坡耕地的1.1-2.3倍、2.0-8.4倍、1.0-2.0倍、1.2-2.4倍.不同粒径团聚体有机碳相比较,大多呈现中间高两边低的变化趋势,最大值出现在中间粒径,即5-2 mm、2-1 mm、1-0.25 mm这3个粒径.逐步回归表明,5-2 mm团聚体和1-0.25 mm团聚体有机碳含量的提高有助于土壤水稳性团聚体的形成.研究结果表明,植被恢复提高了土壤团聚体有机碳含量,在碳形态上,富里酸碳和微生物生物量碳对不同植被恢复措施的敏感度较高,胡敏酸碳含量则相对稳定.     

A microtiter plate assay for polyglutamine aggregate extension

Polyglutamine (polyGln) aggregates are neuropathological markers of expanded CAG repeat disorders, and may also play a critical role in the development of these diseases. We have established a highly sensitive, fast, reproducible, and specific assay capable of monitoring aggregate-dependent deposition of polyglutamine peptides. This assay allows detailed studies on various aspects of aggregation kinetics, and also makes possible the detection and quantitation of low levels of "extension-competent" aggregates. In the simplest form of this assay, polyGln aggregates are made from chemically synthesized peptides and immobilized onto microplate wells. These wells are incubated for different times with low concentrations of a soluble biotinylated polyGln peptide. Europium-streptavidin complexation of the immobilized biotin, followed by time-resolved fluorescence detection of the deposited europium, allows us to calculate the rate (fmol/h) of incorporation of polyGln peptides into polyGln aggregates. This assay will make possible basic studies on the assembly mechanism of polyGln aggregates and on critical features of the reaction, such as polyGln length dependence. The assay also will be a valuable tool for screening and characterizing anti-aggregation inhibitors. It will also be useful for detection and quantitation of aggregation-competent polyGln aggregates in biological materials, which may prove to be of critical importance in understanding the disease mechanism.     

Repeated-batch cultures of Baby Hamster Kidney cell aggregates in stirred vessels

Natural aggregates of Baby Hamster Kidney cells were grown in stirred vessels operated as repeated-batch cultures during more than 600 hours. Different protocols were applied to passaging different fractions of the initial culture: single cells, large size distributed aggregates and large aggregates. When single cells or aggregates with the same size distribution found in culture are used as inoculum, it is possible to maintain semi-continuous cultures during more than 600 hours while keeping cell growth and viability. These results suggest that aggregate culture in large scale might be feasible, since a small scale culture can easily be used as inoculum for larger vessels without noticeable modification of the aggregate chacteristics. However, when only the large aggregates are used as inoculum, it was shown that much lower cell concentrations are obtained, cell viability in aggregates dropping to less than 60%. Under this selection procedure, aggregates maintain a constant size, larger than under batch experiments, up to approximately 400 hours; after this time, aggregate size increases to almost twice the size expected from batch cultures.     

Formation of aggregates by plant roots in homogenised soils

The influence of root growth and water regime on the formation of aggregates was studied in modified minirhizotrons under controlled conditions. Two soils, a black earth (67% clay) and a red-brown earth (19% clay) were ground and forced through a 0.5 mm sieve. Ryegrass, pea and wheat were grown for fifteen wetting and drying (wd) cycles for 5 months. Another set of minirhizotrons was not planted and served as a control. Measurements of aggregate size distribution (ASD), aggregate tensile strength (ATS), aggregate stability (AS), aggregate bulk density (ABD) and organic carbon (OC) were made on single aggregates of the 2–4 mm fraction. The results showed that aggregates of the black earth which has a high clay content and shrink/swell properties had more smaller aggregates with higher ATS, AS and ABD than those from the red-brown earth. It was also found that for both soils: (1) w/d cycles and higher root length density (RLD) increased the proportions of smaller aggregates and aggregate strength; (2) differences in the ability of the plant species to influence aggregation was evident and seemed to be related to the RLD. The RLD was in the order ryegrass > wheat > pea. Mechanisms likely to be involved in processes of aggregate formation and stabilization are discussed. They include cracking of soil due to tensile stresses generated during drying of a shrinking soil; changes in pore water pressure within the soil mass caused by water uptake by plant roots generating effective stresses; and biological processes associated with plant roots and root exudates.     

Isolation and characterization of the hemichrome-stabilized membrane protein aggregates from sickle erythrocytes. Major site of autologous antibody binding

Because the interaction of denatured hemoglobins (i.e. hemichromes) with the red cell membrane has been associated with several abnormalities commonly observed in hemichrome-containing erythrocytes, we have undertaken to isolate and characterize the hemichrome-rich membrane protein aggregates from sickle cells. The aggregates were isolated by two procedures: one at low ionic strength by centrifugation of detergent-solubilized spectrin-depleted inside-out vesicles, and the other at physiological ionic strength by detergent solubilization of whole cells followed by cytoskeletal disruption and centrifugation. The extensively washed aggregates obtained by both methods yielded similar results. These insoluble complexes were found to be highly cross-linked by predominantly intermolecular disulfide bonds; however, other nonreducible covalent linkages were also observed. Both in the presence and absence of reducing agents, the aggregate disintegrated when the hemichromes were removed by high ionic strength, suggesting that the aggregate depended heavily on the cohesive properties of the hemichromes for stability. Protein assays demonstrated that the aggregates comprised approximately 1.3% of the total membrane protein, roughly two-thirds of which appeared to be globin chains. Other major components identified in the aggregate were band 3, ankyrin, bands 4.1, 4.9, and 5, glycophorins A and B, and autologous IgG. Quantitative analysis of the IgG content demonstrated that three-fourths of the surface-bound IgG on washed sickle cells was clustered at these aggregate sites, representing an enrichment of approximately 250-fold over nonaggregated regions of the membrane. Since clustered cell surface IgG is thought to trigger removal of erythrocytes from circulation, the hemichrome-induced membrane reorganization at these aggregate sites may be an important cause of the greatly shortened life span of sickle cells.     

Method validation for the determination of ochratoxin A in green and soluble coffee by immunoaffinity column cleanup and liquid chromatography

A method was validated for the determination of ochratoxin A (OTA) in soluble and green coffee. Performance parameters evaluated included selectivity, accuracy, intermediate precision, linearity, limit of detection, limit of quantitation, and ruggedness. The method was found to be selective for OTA in both matrices tested. Recovery rates from soluble coffee samples ranged from 73.5 to 91.2%, and from green coffee samples from 68.7 to 84.5%. The intermediate precision (RSDr) was between 9.1 and 9.4% for soluble coffee and between 14.3 and 15.5% for green coffee analysis. The linearity of the standard calibration curve (r²) was <0.999 for OTA levels of 1.0–20.0 μg/kg in coffee samples. The limit of detection was determined to be 0.01 ng of OTA on column, while the limit of quantitation was found to be 0.03 ng on column. The limit of quantitation is equivalent to 0.6 μg/kg in soluble coffee samples and 0.3 μg/kg in green coffee samples. The results of the ruggedness trial showed two factors are critical for soluble coffee analysis: the extraction method, and the flow rate of the mobile phase. For green coffee analysis two critical factors detected were the extraction method and the storage temperature of the immunoaffinity column. Five samples of soluble coffee and 42 of green coffee were analysed using the validated method. All soluble coffee samples contained OTA at levels that ranged from 8.4 to 13.9 μg/kg. Six of the 42 green coffee samples analysed (14.3%) contained OTA at levels ranging from 0.9 to 19.4 μg/kg. The validated method can be used to monitor OTA levels in Colombian coffee for export or for local consumption.     

A sensitive HPLC-MS-MS method for the determination of raltegravir in human plasma

This work describes an assay system that has been developed to quantify raltegravir concentrations in human plasma using a liquid-liquid extraction technique paired with HPLC separation and MS-MS detection. The dynamic range of this assay extends from 1 to 3000 ng/mL, with a coefficient of determination (r(2), mean+/-SD) of 0.9992+/-0.0002. The mean precision values for calibration standards ranged from 0.6% to 3.0%, while accuracy values were 96.5-104.3%. This procedure is an accurate, precise, and sensitive method for raltegravir quantitation and was successfully validated using external proficiency testing.     

Determination of laquinimod in plasma by coupled-column liquid chromatography with ultraviolet absorbance detection

Laquinimod is an immunomodulator that is currently in clinical trials. For pharmacokinetic and toxicokinetic studies in animals and humans a sensitive and accurate bioanalytical method was required. In this paper a bioanalytical method for the determination of laquinimod by liquid chromatography is described. After a protein precipitation step the plasma sample was injected onto a coupled-column HPLC system. After further purification from macromolecules on a short restricted access material C(18) column the analyte was transferred to a reversed-phase C(18) analytical column and separated from interfering substances. The analyte was detected by UV detection. The method was validated with respect to linearity, selectivity, precision, accuracy, limit of quantitation, limit of detection, recovery and stability. The limit of quantitation was 0.75 micromol/L, the intermediate precision was 1.8-3.6% (C.V.) and the accuracy was 97.7-114.7%. In conclusion, the method was found to perform well and is suitable for use in pharmacokinetic and toxicokinetic studies.     

Development and validation of chiral high-performance liquid chromatographic methods for the quantitation of valsartan and of the tosylate of valinebenzyl ester

A stereospecific HPLC method for the quantitation of CGP 49309 in samples of its corresponding enantiomer valsartan has been developed and validated. The enantiomeric separation was achieved on a 5 μm silica-bonded α₁-acid glycoprotein column (Chiral AGP) with a phosphate buffer, pH 7, containing 2% (v/v) 2-propanol as a mobile phase. The linearity was established in the range 0.1–4% (r>;0.999). The limit of quantitation was 0.1% and the limit of detection was 0.04%. The accuracy of the method was found to be 96.7% (average). For the precision (repeatability), a relative standard deviation value of 2.4% was found. Similarly, a stereoselective HPLC method was also developed and validated for the quantitation of the enantiomer of the starting material used for the synthesis of valsartan, namely (R)-valinebenzyl ester tosylate. Baseline resolution of the enantiomers of valinebenzyl ester tosylate could be achieved on the chiral crown ether column Crownpak CR (Daicel) at 50°C using water-methanol-trifluoroacetic acid (850:150:1, v/v) as a mobile phase. The linearity was established in the range 0.5-5% (r>;0.999). The accuracy of the method was found to be 100.5% (average). For the precision (repeatability), a relative standard deviation value of 3.4% was found. Both methods were found to be suitable for the analysis of the respective analytes.     

Physicochemical studies on goat pulmonary surfactant

The large aggregate (LA) fraction of goat pulmonary surfactant (GPS) was isolated and characterized. Goat lung surfactant extract (GLSE) was obtained by chloroform-methanol extraction of the saline suspended LA fraction. Total phospholipid (PL), cholesterol (CHOL), and protein were biochemically estimated. It was composed of approximately 83% (w/w) PL, approximately 0.6% (w/w) CHOL and approximately 16% (w/w) protein. CHOL content was found to be lower while the protein content was found to be higher than other mammalian pulmonary surfactants. Electrospray Ionization Mass Spectrometry (ESIMS) of GLSE confirmed dipalmitoylphosphatidylcholine (DPPC) as the major phospholipid species, with significant amounts of palmitoyl-oleoyl phosphatidylcholine (POPC), palmitoyl-myristoyl phosphatidylcholine (PMPC) and dioleoylphosphatidylcholine (DOPC). Functionality of the solvent spread GLSE film was carried out in a Langmuir surface balance by way of surface pressure (pi)-area (A) measurements. A high value of pi (approximately 65 mN m(-1)) could be attained with a lift-off area of approximately 1.2 nm(2) molecule(-1). A relatively large hysteresis was observed during compression-expansion cycles. Monolayer deposits at different pi, transferred onto freshly cleaved mica by Langmuir-Blodgett (LB) technique, were imaged by atomic force microscopy. DPPC-enriched domains (evident from height analyses) showed dimensions of 2.5 microm and underwent changes in shapes after 30 mN m(-1). Functionality and structure of the surfactant films were proposed to be controlled by the relative abundances of protein and cholesterol.     

Evaluation of capillary supercritical fluid chromatography with mass spectrometric detection for the analysis of a drug (mebeverine) in a dog plasma matrix

Supercritical fluid chromatography with mass spectrometric detection was evaluated as a technique for the analysis of drugs in biological fluids. Dog plasma was spiked with a model drug, mebeverine, and with a deuterium-labeled analog of mebeverine. The spiked plasma was prepared for analysis by solid-phase extraction on octadecylsilane cartridges. Mebeverine levels in the spiked dog plasma samples were determined by interpolation from a standard curve. Accuracy and precision of the analysis were determined within and between days. In general, accuracy was found to be 100 ± 15% for plasma samples spiked with 6 to 60 ng mebeverine/ml. The relative standard deviation for replicate sample analysis over this concentration range was between 5 and 12.5%.     

Scale-up of breast cancer stem cell aggregate cultures to suspension bioreactors

It has been hypothesized that breast tumor formation results from the activity of a scarce population of cells known as Breast Cancer Stem Cells (BrCSCs) and that the development of effective breast cancer therapies may therefore ultimately rely upon the ability to effectively target these cells for eradication. The scarcity of BrCSCs in vivo severely compromises research on these populations, as analyses are restricted to those requiring small cell numbers, and has become a major impediment to the development of therapeutic strategies against breast cancer. Through the culture of murine tissue aggregates containing a population of BrCSCs, this study demonstrates the ability of propagating this scarce population in a controlled and reproducible manner, within suspension bioreactors. A rigorous theoretical framework has been developed in order to understand and characterize the implications of oxygen mass transfer within aggregates upon scale-up and thereby provide a foundation for the scale-up of aggregate cultures. A two-factor, two-level factorial experimental design was also performed in order to assess the effects of inoculation density and hydrodynamic shear upon cell yield. We discovered that the culture of the murine aggregates in a relatively low shear environment (tau(max) = 0.20 Pa) and inoculated at 3.50 x 10(4) cells/mL resulted in the best yields for the range of conditions investigated in suspension bioreactors. A detailed study on the oxygen uptake kinetics of the aggregates also revealed that the uptake rates were not significantly affected by mass transfer limitations, as uptake rates of aggregate cultures were found to be comparable to those observed in single cell cultures. Cells propagated in a process controlled 500 mL suspension bioreactor resulted in growth kinetics that were comparable to those observed in 125 mL bioreactors. Doubling times in the 500 mL vessel were found to be 23.9 h and attained a maximum cell density of 1.20 x 10(6) cells/mL. After enumerating the number of BrCSCs, this resulted in an approximately 20-fold increase in BrCSC numbers in batch suspension cultures. With greater attention being applied to BrCSCs, their propagation in suspension bioreactors makes available experimental avenues that are not currently accessible and may thereby enable the development of more effective therapeutic drugs for the treatment of breast cancer.     

Direct determination of polyglucose metabolites in plasma using anion-exchange chromatography with pulsed amperometric detection

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE–PAD) was evaluated for the quantitation of polyglucose metabolites (DP2–DP7) in human plasma. The method was investigated for accuracy, precision, specificity, linearity, range and analyte stability. Samples were prepared by dilution into the standard range (0.1–10 μg/ml) followed by deproteinization using a 30?000 molecular mass cut-off filtration device. The limit of detection was 0.05 μg/ml for all metabolites. Method precision for DP2–DP7 varied from approximately 2% R.S.D. in the upper range to approximately 15% R.S.D. at the limit of quantitation. Samples were stable following one or two freeze–thaw cycles and, after preparation, they could be refrigerated for up to 72 h. Application of this method to clinical plasma samples from continuous ambulatory peritoneal dialysis (CAPD) patients administered one daily night-time intraperitoneal exchange of 2 l of 7.5% polyglucose solution for four weeks indicated that plasma levels of DP2, DP3 and DP4 increased from baseline levels of <0.01 g/l to steady-state levels of 1.2±0.3, 1.2±0.3 and 0.4±0.1 g/l (mean±S.D.), respectively. These steady state plasma levels for DP2 and DP3 are comparable to previously reported levels in patients administered daily overnight 7.5% polyglucose dialysis solution.     

Development of an RSA calibration system with improved accuracy and precision

In this study, a new radiostereometric analysis (RSA) calibration cage was developed with the aim of improving the accuracy and precision of RSA. This development consisted of three steps: a numerical simulation technique was first used to design the new cage; a synthetic imaging method was then implemented to predict the performance of the designed cage before it was actually fabricated; and an experimental phantom test was finally conducted to verify the actual performance of the new cage and compare with two currently widely used cages. Accuracy was calculated as the 95% prediction intervals from regression analyses between the measured and actual displacements, and precision was defined as the standard deviation of repeated measurements. The final experimental phantom tests showed that the accuracy and precision of the new calibration cage were improved by about 40% over an existing biplanar cage and by about 70% compared to a uniplanar cage design. This new cage can be used with any skeletal joints, in either static or kinematic examination, which is helpful for the standardization of the RSA application.     

Analysis of cocaethylene,benzoylecgonine and cocaine in human urine by high-performance thin-layer chromatography with ultraviolet detection: a comparison with high-performance liquid chromatography

Cocaine and ethanol are frequently used at the same time, resulting in the formation of cocaethylene by transesterification. We studied the capability of high-performance thin-layer chromatography (HPTLC) to simultaneously detect cocaethylene, cocaine and benzoylecgonine in 16 urine specimens of drug addicts, previously tested as positive for benzoylecgonine at immunoenzymatic screening. Accuracy and precision, as well as detection and quantitation limits of the method, were evaluated by comparison with high-performance liquid chromatography (HPLC). HPTLC limit of quantitation was 1.0 μg/ml for the three compounds, whereas HPLC limits were 0.2 μg/ml for benzoylecgonine and cocaine, and 0.1 μg/ml for cocaethylene. The relative standard deviation (RSD) ranged from 1.03 to 12.60% and from 1.56 to 16.6% for intra- and inter-day HPTLC analysis, respectively. In the case of the HPLC method, the RSD for the intra-day precision ranged from 0.79 to 5.05%, whereas it ranged from 1.19 to 10.64% for the inter-day precision. In comparison with HPLC, HPTLC is less expensive and faster, requiring 2–3 h to analyze 10–12 samples on a single plate. In conclusion, HPTLC is suitable for determinations of the three analytes only for samples with high concentrations.     

Determination of selenium in urine by isotope dilution gas chromatography-mass spectrometry using 4-nitro-o-phenylenediamine, 3,5-dibromo-o-phenylenediamine, and 4-trifluoromethyl-o-phenylenediamine as derivatizing reagents.

An isotope dilution gas chromatography-mass spectrometry method for selenium determination in urine using 76Se as an internal standard is described. Three different derivatizing reagents, 4-nitro-o-phenylenediamine (NPD), 3,5-dibromo-o-phenylenediamine (DBPD), and 4-trifluoromethyl-o-phenylenediamine (TFMPD) were investigated for their gas chromatographic behavior including memory and precision and accuracy of isotope ratio measurements. By these criteria, the performance of these reagents was TFMPD greater than DBPD greater than NPD. Overall precision values of 1 to 7% were observed in determining Se isotope ratios at the 10-ng level, with no significant difference in using any of the three reagents. Memory effect was observed in the order NPD greater than DBPD greater than TFMPD with TFMPD showing no measurable memory effect. Accuracy of the GC-MS method was verified by quantitation of selenium in the NIST freeze-dried urine reference material SRM 2670.     

High-performance liquid chromatographic assay for minocycline in human plasma and parotid saliva

A sensitive and specific high-performance liquid chromatographic (HPLC) method with UV detection was developed for the determination of minocycline in human plasma and parotid saliva samples. Samples were extracted using an Oasis™ HLB cartridge and were injected into a C₈ Nucleosil column. The HPLC eluent contained acetonitrile–methanol–distilled water–0.1% trifluoroacetic acid (25:2:72.9:0.1, v/v). Demeclocycline was used as internal standard. The assay showed linearity in the tested range of 0.1–25 μg/ml. The limit of quantitation was 100 ng/ml. Recovery from plasma or parotid saliva averaged 95%. Precision expressed as %CV was in the range 0.2–17% (limit of quantitation). Accuracy ranged from 93 to 111%. In the two matrices studied at 20 and 4°C, rapid degradation of the drug occurred. Frozen at −30°C, this drug was stable for at least 2 months, the percent recovery averaged 90%. The method’s ability to quantify minocycline with precision, accuracy and sensitivity makes it useful in pharmacokinetic studies.     

Simultaneous determination of 8 HIV protease inhibitors in human plasma by isocratic high-performance liquid chromatography with combined use of UV and fluorescence detection: amprenavir, indinavir, atazanavir, ritonavir, lopinavir, saquinavir, nelfinavir and M8-nelfinavir metabolite

A simple, accurate and fast method was developed for determination of the commonly used HIV protease inhibitors (PIs) amprenavir, indinavir, atazanavir, ritonavir, lopinavir, nelfinavir, M8-nelfinavir metabolite and saquinavir in human plasma. Liquid-liquid extraction was used with hexane/ethylacetate from buffered plasma samples with a borate buffer pH 9.0. Isocratic chromatographic separation of all components was performed on an Allsphere hexyl HPLC column with combined UV and fluorescence detection. Calibration curves were constructed in the range of 0.025-10 mg/l. Accuracy and precision of the standards were all below 15% and the lowest limit of quantitation was 0.025 mg/l. Stability of quality control samples at different temperature conditions was found to be below 20% of nominal values. The advantages of this method are: (1) inclusion and determination of the newly approved atazanavir, (2) simultaneous isocratic HPLC separation of all compounds and (3) increased specificity and sensitivity for amprenavir by using fluorescence detection. This method can be used for therapeutic drug monitoring of all PIs currently commercialised and is now part of current clinical practice.