Simultaneous LC-MS/MS bioanalysis of etoposide and paclitaxel in mouse tissues and plasma after oral administration of self-microemulsifying drug-delivery systems.

In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine the anticancer drugs etoposide and paclitaxel in mouse plasma and tissues including liver, kidney, lung, heart, spleen and brain. The analytes were extracted from the matrices of interest by liquid-liquid extraction using methyl tert-butyl ether-dichloromethane (1:1, v/v). Chromatographic separation was achieved on an Ultimate XB-C18 column (100 × 2.1 mm, 3 µm) at 40°C and the total run time was 4 min under a gradient elution. Ionization was conducted using electrospray ionization in the positive mode. Stable isotope etoposide-d3 and docetaxel were used as the internal standards. The lower limit of quantitation (LLOQ) of etoposide was 1 ng/g tissue for all tissues and 0.5 ng/mL for plasma. The LLOQ of paclitaxel was 0.4 ng/g tissue and 0.2 ng/mL for all tissues and plasma, respectively. The coefficients of correlation for all of the analytes in the tissues and plasma were >0.99. Both intra- and inter-day accuracy and precision were satisfactory. This method was successfully applied to measure plasma and tissue drug concentrations in mice treated with etoposide and paclitaxel-loaded self-microemulsifying drug-delivery systems.

Electrochemical sensing of etoposide using carbon quantum dot modified glassy carbon electrode.

In this study, enhancement of the electrochemical signals of etoposide (ETO) measured by differential pulse voltammetry (DPV) by modifying a glassy carbon electrode (GCE) with carbon quantum dots (CQDs) is demonstrated. In comparison with a bare GCE, the modified GCE exhibited a higher sensitivity towards electrochemical detection of ETO. The lowest limit of detection was observed to be 5 nM ETO. Furthermore, scanning electron microscopy (SEM), fluorescence microscopy (FM), and electrochemical impedance spectroscopy (EIS) were employed for the further study of the working electrode surface after the modification with CQDs. Finally, the GCE modified with CQDs under optimized conditions was used to analyse real samples of ETO in the prostate cancer cell line PC3. After different incubation times (1, 3, 6, 9, 12, 18 and 24 h), these samples were then prepared prior to electrochemical detection by the GCE modified with CQDs. High performance liquid chromatography with an electrochemical detection method was employed to verify the results from the GCE modified with CQDs.

Needle-in-needle electrochemical sensor for in-vivo monitoring of anticancer drug etoposide.

Therapeutic drug monitoring (TDM) serves as a potent tool for adjusting drug concentration within a reasonable range. However, continuous monitoring of anticancer drugs in-vivo presents a significant challenge. Herein, we propose a needle-in-needle electrochemical sensor based on an acupuncture needle electrode, capable of monitoring the anticancer drug etoposide in the peritoneal cavity of living rats. The acupuncture needle was modified with Au nanoparticles and etoposide-templated molecularly imprinted polymer (MIP), resulting in high sensitivity and selectivity in the electrochemical detection of etoposide. The modified acupuncture needle (0.16 mm diameter) was anchored inside a syringe needle (1.40 mm diameter), allowing the outer syringe needle to protect the modified materials of the inner acupuncture needle during skin piercing. Due to the unique needle-in-needle design, high stability was obtained during in-vivo etoposide monitoring. Connecting to a smartphone-controlled portable electrochemical workstation, the needle-in-needle sensor offers great convenience in point-of-care TDM. Moreover, the electrode materials on the acupuncture needle were carefully characterized and optimized. Under the optimized conditions, low detection limits and wide linear range were achieved. This work provides new insights into acupuncture needle electrochemical sensors and further expands the feasibility for real-time and in-vivo detection.

Multi-target analysis of cytostatics in hospital effluents over a 9-month period.

The consumption of cytostatics, pharmaceuticals prescribed in chemotherapy, is increasing every year and worldwide, along with the incidence of cancer. The presence and the temporal evolution of cytostatics in wastewaters from a Portuguese hospital center was evaluated through a 9-month sampling campaign, comprising a total of one hundred and twenty-nine samples, collected from May 2019 to February 2020. Eleven cytostatics out of thirteen pharmaceuticals were studied, including flutamide, mycophenolate mofetil and mycophenolic acid, which have never been monitored before. Target analytes were extracted and quantified by solid-phase extraction coupled to liquid-chromatography-tandem mass spectrometry analysis; the method was fully validated. All pharmaceuticals were detected in at least one sample, bicalutamide being the one found with higher frequency (detected in all samples), followed by mycophenolic acid, which was also the compound detected at higher concentrations (up to 5340 ± 211 ng/L). Etoposide, classified as carcinogenic to humans, was detected in 60% of the samples at concentrations up to 142 ± 15 ng/L. The risk from exposure to cytostatics was estimated for aquatic organisms living in receiving bodies. Cyclophosphamide, doxorubicin, etoposide, flutamide, megestrol and mycophenolic acid are suspected to induce risk. Long-term and synergic effects should not be neglected, even for the cytostatics for which no risk was estimated.

LC-MS-MS Determination of Cytostatic Drugs on Surfaces and in Urine to Assess Occupational Exposure.

The ever-increased usage of cytostatic drugs leads to high risk of exposure among healthcare workers. Moreover, workers are exposed to multiple compounds throughout their lives, leading to cumulative and chronic exposure. Therefore, multianalyte methods are the most suitable for exposure assessment, which minimizes the risks from handling cytostatic drugs and ensures adequate contamination containment. This study describes the development and full validation of two liquid chromatography-tandem mass spectrometry methods for the detection of gemcitabine, dacarbazine, methotrexate, irinotecan, cyclophosphamide, doxorubicinol, doxorubicin, epirubicin, etoposide, vinorelbine, docetaxel and paclitaxel in working surfaces and urine samples. The urine method is the first to measure vinorelbine and doxorubicinol. For surfaces, limits of detection (LOD) and limits of quantification (LOQ) were 5-100 pg/cm2, and linearity was achieved up to 500 pg/cm2. Inaccuracy was between -11.0 and 8.4%. Intra-day, inter-day and total imprecision were <20%, except for etoposide and irinotecan (<22.1%). In urine, LOD and LOQ were 5-250 pg/mL, with a linear range up to 1,000-5,000 pg/mL. Inaccuracy was between -3.8 and 14.9%. Imprecision was <12.4%. Matrix effect was from -58.3 to 1,268.9% and from -66.7 to 1,636% in surface and urine samples, respectively, and extraction efficiency from 10.8 to 75% and 47.1 to 130.4%, respectively. All the analytes showed autosampler (6°C/72 h), freezer (-22°C/2 months) and freeze/thaw (three cycles) stability. The feasibility of the methods was demonstrated by analyzing real working surfaces and patients' urine samples. Contamination with gemcitabine, irinotecan, cyclophosphamide, epirubicin and paclitaxel (5-4,641.9 pg/cm2) was found on biological safety cabinets and outpatients' bathrooms. Analysis of urine from patients under chemotherapy identified the infused drugs at concentrations higher than the upper LOQ. These validated methods will allow a comprehensive evaluation of both environmental and biological contamination in hospital settings and healthcare workers.

Wipe sampling procedure coupled to LC-MS/MS analysis for the simultaneous determination of 10 cytotoxic drugs on different surfaces.

A simple wipe sampling procedure was developed for the surface contamination determination of ten cytotoxic drugs: cytarabine, gemcitabine, methotrexate, etoposide phosphate, cyclophosphamide, ifosfamide, irinotecan, doxorubicin, epirubicin and vincristine. Wiping was performed using Whatman filter paper on different surfaces such as stainless steel, polypropylene, polystyrol, glass, latex gloves, computer mouse and coated paperboard. Wiping and desorption procedures were investigated: The same solution containing 20% acetonitrile and 0.1% formic acid in water gave the best results. After ultrasonic desorption and then centrifugation, samples were analysed by a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in selected reaction monitoring mode. The whole analytical strategy from wipe sampling to LC-MS/MS analysis was evaluated to determine quantitative performance. The lowest limit of quantification of 10 ng per wiping sample (i.e. 0.1 ng cm(-2)) was determined for the ten investigated cytotoxic drugs. Relative standard deviation for intermediate precision was always inferior to 20%. As recovery was dependent on the tested surface for each drug, a correction factor was determined and applied for real samples. The method was then successfully applied at the cytotoxic production unit of the Geneva University Hospitals pharmacy.

Focused Ultrasound-Mediated Blood-Brain Barrier Opening Increases Delivery and Efficacy of Etoposide for Glioblastoma Treatment.

PURPOSE: Glioblastoma (GBM) is a devastating disease. With the current treatment of surgery followed by chemoradiation, outcomes remain poor, with median survival of only 15 months and a 5-year survival rate of 6.8%. A challenge in treating GBM is the heterogeneous integrity of the blood-brain barrier (BBB), which limits the bioavailability of systemic therapies to the brain. There is a growing interest in enhancing drug delivery by opening the BBB with the use of focused ultrasound (FUS). We hypothesize that an FUS-mediated BBB opening can enhance the delivery of etoposide for a therapeutic benefit in GBM. METHODS AND MATERIALS: A murine glioma cell line (Pdgf+, Pten-/-, P53-/-) was orthotopically injected into B6(Cg)-Tyrc-2J/J mice to establish the syngeneic GBM model for this study. Animals were treated with FUS and microbubbles to open the BBB to enhance the delivery of systemic etoposide. Magnetic resonance (MR) imaging was used to evaluate the BBB opening and tumor progression. Liquid chromatography tandem mass spectrometry was used to measure etoposide concentrations in the intracranial tumors. RESULTS: The murine glioma cell line is sensitive to etoposide in vitro. MR imaging and passive cavitation detection demonstrate the safe and successful BBB opening with FUS. The combined treatment of an FUS-mediated BBB opening and etoposide decreased tumor growth by 45% and prolonged median overall survival by 6 days: an approximately 30% increase. The FUS-mediated BBB opening increased the brain tumor-to-serum ratio of etoposide by 3.5-fold and increased the etoposide concentration in brain tumor tissue by 8-fold compared with treatment without ultrasound. CONCLUSIONS: The current study demonstrates that BBB opening with FUS increases intratumoral delivery of etoposide in the brain, resulting in local control and overall survival benefits.

Target specific intracellular quantification of etoposide by quadrupole-time of flight based mass spectrometric method.

Etoposide (ETP), a widely used chemotherapeutic agent has an intracellular target site of action. Unfortunately, the concentration of ETP in plasma does not properly reflect the concentration in its intracellular site of action. As per our knowledge, no reported bioanalytical method is available for intracellular quantification of ETP. In this research, we developed an LC-MS/MS method to quantitate ETP in intracellular compartments of MCF-7 cells. The Abcam nuclear extraction kit was used for extracting the nuclear and cytosolic protein from MCF-7 cells. The method showed excellent linearity in the 20-1000 ng/mL range. The intra and inter-day precision (%CV) including LLOQ were found to be in the range of 2.19-16.96% and 6.71-11.21%, respectively, with an accuracy of 86.87 to 110.37% and 93.03 to 100.50%, respectively. The concentration of ETP in nuclear and cytosolic fraction was successfully quantitated using the developed method. The developed method can be applied to understand the efficacy of different formulations based on the intracellular ETP concentration in vitro. It can be considered as a model method for quantification of other similar categories of drugs in their actual intracellular site of action after required optimization in the methodology.

Determination of teniposide in rat plasma by ultra performance liquid chromatography electrospray ionization tandem mass spectrometry after intravenous administration.

A novel, specific and rapid ultra performance liquid chromatography electrospray ionization tandem mass spectrometry method has been developed and validated for determination of teniposide in rat plasma. A one-step liquid-liquid extraction method was used and the separation was carried out on an Acquity UPLC(TM) BEH C(18) column with gradient elution using a mobile phase composed of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.20 mL/min. A triple quadrupole tandem mass spectrometer in multiple-reaction monitoring mode via an electrospray ionization interface was used for the detection of teniposide. The detection was complete within 3.0 min. A linear calibration curve was obtained over the concentration range 10-10,000 ng/mL for teniposide, with a lower limit of quantification of 10 ng/mL. The intra-day precision and inter-day precision (relative standard deviation) were less than 10.23 and 13.09%, respectively. The developed method was applied for the first time to the pharmacokinetic study of teniposide in rats following a single intravenous administration of 4.5 mg/kg teniposide.

Toxicological study of the degradation products of antineoplastic agent etoposide in commercial formulation treated by heterogeneous photocatalysis using SrSnO3.

Etoposide is an antineoplastic agent used for treating lung cancer, testicular cancer, breast cancer, pediatric cancers, and lymphomas. It is a pollutant due to its mutagenic and carcinogenic potential. Disposal of waste from this drug is still insufficiently safe, and there is no appropriate waste treatment. Therefore, it is important to use advanced oxidative processes (AOPs) for the treatment and disposal of medicines like this. The use of strontium stannate (SrSnO3) as a catalyst in heterogeneous photocatalysis reactions has emerged as an alternative for the removal of organic pollutants. In our study, SrSnO3 was synthesized by the combustion method and characterized by X-ray diffraction (XRD), Raman, UV-Vis, and scanning electron microscopy (SEM) techniques, obtaining a surface area of 3.28 m2 g-1 with cubic and well-organized crystallinity and a band gap of 4.06 eV. The experimental conditions optimized for degradation of an etoposide solution (0.4 mg L-1) were pH 5 and catalyst concentration of 1 g L-1. The results showed that the degradation processes using SrSnO3 combined with H2O2 (0.338 mol L-1) obtained total organic carbon removal from the etoposide solution, 97.98% (± 4.03 × 10-3), compared with TiO2, which obtained a mineralization rate of 72.41% (± 6.95 × 10-3). After photodegradation, the degraded solution showed no toxicity to zebrafish embryos through embryotoxicity test (OECD, 236), and no genotoxicity using comet assay and micronucleus test.

Au/Pd@rGO nanocomposite decorated with poly (L-Cysteine) as a probe for simultaneous sensitive electrochemical determination of anticancer drugs, Ifosfamide and Etoposide.

The simultaneous measurement of the concentration of anticancer drugs with a fast, sensitive and accurate method in biological samples is a challenge for better monitoring of drug therapy and better determine the pharmacokinetics. An electrochemical sensor was developed for the simultaneous determination of anticancer drugs, Ifosfamide (IFO) and Etoposide (ETO) based on pencil graphite electrode modified with Au/Pd@rGO nanocomposite decorated with poly (L-Cysteine). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were utilized to study the properties of the modified electrode. The electrochemical behavior of IFO and ETO on the Au/Pd@rGO@p(L-Cys) modified electrode was investigated by cyclic voltammetry and differential pulse voltammetry (DPV) techniques and the obtained results confirmed its efficiency for the individual and simultaneous sensing of IFO and ETO. After optimization of electrochemical parameters, the fabricated sensor presented excellent performance in simultaneous determination of IFO and ETO with a wide linear range from 0.10 to 90.0 µM and 0.01 to 40.0 µM and low detection limits (3 Sb/m) of 9.210 nM and 0.718 nM, respectively. In addition, this study proved that the constructed sensor could be useful to simultaneous analysis of IFO and ETO in biological samples and pharmaceutical compounds.

New, sensitive and validated spectrofluorimetric method for the estimation of etoposide in bulk and pharmaceutical formulations.

A new, simple and sensitive spectrofluorimetric method was developed for the routine estimation of etoposide, an anticancer drug, in bulk and in pharmaceutical formulations. The medium selected for the estimation of etoposide was methanol : phosphate buffer (pH 7.4) in a ratio of 70 : 30 v/v. Excitation and emission wavelengths used were 240 and 324 nm respectively. The method was validated according to ICH and U.S.P guidelines. Linearity range was 200-1000 ng/ml with detection and quantitation limits of 23 ng/ml and 72 ng/ml respectively. Regression equation obtained was Fluorescence Intensity = 6.704 (Concentration in ng/ml) + 115.4. The drug was found to be stable and there was no interference from the excipients present in different formulations of etoposide.

A selective and sensitive stability-indicating HPLC method for the validated assay of etoposide from commercial dosage form and polymeric tubular nanocarriers.

Etoposide is a topoisomerase II enzyme inhibitor type chemotherapeutic agent which is widely used in the therapy of various cancers. Its short half-life and toxicity to normal tissues are the major drawbacks in its clinical applications. Polymeric nanoparticulate drug delivery systems are rational carriers to deliver etoposide with higher efficiency and fewer side effects. In addition tubular shaped drug carriers are found to show a great potential for drug delivery on the basis of promising results regarding particle shape and cellular uptake. In this study, etoposide loaded polymeric tubular nanocarriers have been developed by template wetting method using porous anodic aluminum oxide membranes as templates. The developed poly(methyl methacrylate) nanocarriers were evaluated for structural analysis, in vitro drug release studies and drug release kinetics. Accurate and reliable determination of the drug release from newly developed nanocarriers, is of great importance. For this reason a selective and sensitive reversed phase liquid chromatography method was developed and fully validated from the point of system suitability, specificity, linearity and range, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and robustness for the reliable determination of etoposide. Stability indicating capability was shown with forced degradation studies and the chromatographic conditions were optimized on ACE 5C18 (150 mm × 4.6mm I.D., 5 µm) analytical column. Related to the calibration results ETP was found linear in the range between 0.2 from 100 µg mL(-1) with the LOD as 0.015 µg.mL(-1). The resultant conditions were applied for the selective and sensitive determination of etoposide from its commercial dosage form with the high accuracy values (99.82-100.65%). The method was successfully detected assay of etoposide release from newly developed polymeric tubular nanocarriers, which was found as 72.2% at the end of 24h.

Etoposide-loaded nanoparticles made from glyceride lipids: formulation, characterization, in vitro drug release, and stability evaluation.

The aim of the study was to prepare etoposide-loaded nanoparticles with glyceride lipids and then characterize and evaluate the in vitro steric stability and drug release characteristics and stability. The nanoparticles were prepared by melt emulsification and homogenization followed by spray drying of nanodispersion. Spray drying created powder nanoparticles with excellent redispersibility and a minimal increase in particle size (20-40 nm). Experimental variables, such as homogenization pressure, number of homogenization cycles, and surfactant concentration, showed a profound influence on the particle size and distribution. Spray drying of Poloxamer 407-stabilized nanodispersion lead to the formation of matrix-like structures surrounding the nanoparticles, resulting in particle growth. The in vitro steric stability test revealed that the lipid nanoparticles stabilized by sodium tauroglycocholate exhibit excellent steric stability compared with Poloxamer 407. All 3 glyceride nanoparticle formulations exhibited sustained release characteristics, and the release pattern followed the Higuchi equation. The spray-dried lipid nanoparticles stored in black polypropylene containers exhibited excellent long-term stability at 25 degrees C and room light conditions. Such stable lipid nanoparticles with in vitro steric stability can be a beneficial delivery system for intravenous administration as long circulating carriers for controlled and targeted drug delivery.

Degradation of the cytostatic etoposide in chlorinated water by liquid chromatography coupled to quadrupole-Orbitrap mass spectrometry: identification and quantification of by-products in real water samples.

Once discharged into the sewage system, many pharmaceuticals may undergo degradation reactions in the presence of chemical disinfectants, generating by-products that may possess enhanced toxicity relative to the parent compounds. For this reason, the stability of the widely used cytostatic etoposide in chlorinated water has been investigated for the first time in the present work. Taking advantage of the high-resolution/accurate-mass capabilities of the hybrid quadrupole-Orbitrap mass spectrometer Q Exactive, two new oxidation by-products of etoposide were reliably identified. The time course of etoposide and its by-products was followed at different pH values, free chlorine concentrations and water matrices. Finally, the occurrence of etoposide and its major identified by-product (3'-O-desmethyl etoposide) was investigated in real water samples by on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry using a 4000QTRAP hybrid quadrupole-linear ion trap mass spectrometer. The etoposide by-product was found in various river and wastewater samples at levels between 14 and 33 ng L(-1), whereas etoposide was not detected in any sample.

Radioimmunoassay for etoposide and teniposide.

A radioimmunoassay for VP-16 or VM-26 was developed by using tritiated ligand and antisera produced from rabbits immunized with succinyl-VP-16 bovine serum albumin conjugates. Separate determinations of VP-16 and its hydroxy acid, a metabolite which cross-reacted with the VP-16 antisera, could be accomplished by extracting samples with chloroform in which the metabolite was insoluble. The assay was reproducible and sensitive. Extracted standard curves were linear from 0.025 to 5 micrograms for VP-16 and 0.1 to 10 micrograms for the hydroxy acid per 0.5 ml assay mixture. Fifty percent inhibition of binding was achieved at 0.066 and 0.55 microgram for VP-16 or VM-26 and the metabolite, respectively. Preliminary disposition studies in mice and dog, and human urinary excretion support the application of the assay in pharmacologic studies.

Immunocytochemical localisation of VP16-213 in normal and malignant tissues.

Immunocytochemistry has been used to visualise the distribution of the anti-cancer drug VP16-213 in tissues from normal and leukaemic mice, and in EMT6 mammary tumours. Uptake of the drug into EMT6 tumour tissue was poor compared to uptake into normal tissues (liver, kidney, heart, small intestine). Uptake of the drug into tissues from L1210 mice was greater than that into normal tissues. Immunocytochemistry promises to be a very useful additional technique for studying the distribution of drugs given singly or in combination, specific organ toxicities, and mechanisms of resistance.

DNA damage induced by etoposide; a comparison of two different methods for determination of strand breaks in DNA.

Etoposide induces DNA damage to cells by interacting with the nuclear enzyme topoisomerase II. In this investigation the human lymphoblastic leukemia cell line (CEM) was used to study induction of DNA-strand breaks and cellular drug uptake after treatment with etoposide at a concentration of 0.5-2 micrograms/ml. High performance liquid chromatography was used for determination of etoposide concentrations. The alkaline elution assay and the DNA unwinding technique were compared for quantifying strand breaks in DNA induced by etoposide. The concentrations required to increase the level of DNA damage significantly was as follows: the DNA unwinding technique, 0.20 microgram/ml; the alkaline elution assay with proteinase K, 0.45 microgram/ml; the alkaline elution assay without proteinase K, 0.60 microgram/ml. When the half-life was adjusted, considering the efflux time of etoposide from cells, it was found to be only a few minutes. The present data show that the DNA unwinding technique is to be preferred for the screening of DNA damage. This technique is easier and quicker to perform than the alkaline elution technique.

Effect of tricaprin on the physical characteristics and in vitro release of etoposide from PLGA microspheres.

The purpose of this article is to examine the effects of tricaprin on the physical characteristics and in vitro release of etoposide from poly (lactic-co-glycolic acid) microspheres. The microspheres were synthesized through the use of a single-emulsion solvent-extraction procedure. Samples from each batch of microspheres were then analyzed for size distribution, drug loading efficiency, surface characteristics, in vitro release, and in vitro degradation of microspheres. Microsphere batches were synthesized using three different etoposide concentrations (15%, 10%, and 5% w/w) with tricaprin concentrations of 25% and 50%. The incorporation of 50% tricaprin significantly increased (p<0.05) the size of the microspheres for all three etoposide concentrations in comparison to microspheres prepared without tricaprin (control). The percentage of tricaprin used did not significantly affect the drug loading efficiency of the microspheres. The addition of tricaprin was shown to significantly increase (p<0.05) the in vitro release of etoposide from the microspheres prepared with all three concentrations of etoposide and the two different tricaprin percentages. Examination of the surface characteristics of the tricaprin loaded microspheres showed a dimpled surface with what appeared to be pockets of tricaprin dispersed throughout. In the in vitro degradation study, the tricaprin microspheres grew very porous as the degradation time increased, but they still retained a recognizable structure even after 30 days of degradation.

Drug monitoring of etoposide (VP16-213). I. A combined method of liquid chromatography and mass spectrometry.

Drug monitoring is performed by means of sample extraction, sample purification by high-performance liquid chromatography (HPLC), and sample detection by time-of-flight mass spectrometry. This mass spectrometry utilizing 252Cf fission fragment-induced ionization and desorption of nonvolatile compounds is suitable as a universal, nondestructive detector in HPLC. Liquid chromatography and mass spectrometry are combined, so that mass analysis can be operated online and offline to the fractional sampling of the effluent and the samples can still be recovered. As an alternative to HPLC separation, samples can be purified by thin-layer chromatography (TLC), resulting an offline TLC + MS combination. Preliminary pharmacokinetic data for etoposide (VP16-213) together with calibration data are presented, and are discussed with reference to the sensitivity and detection limit of the new experimental method.