28 minute read
Pharma Notes
Bioniche Life Sciences
Inc. (Belleville, ON) has entered into two distribution agreements to expand its product offerings in North America. The first agreement is with MedTrade Products Limited, a UK-based company. The agreement provides a number of equine and companion animal health products based on MedTrade’s CeloxTM technology for exclusive distribution by Bioniche in Canada. The CeloxTM technology uses chitosan, a natural polysaccharide, as a medical device in the animal health industry. The CeloxTM product line includes gauzes and granules that are utilized in wound healing. When mixed with blood, CeloxTM forms a gel-like clot in less than one minute, independent of the body’s normal clotting processes. The second agreement is with Mueller Medical International LLC, a U.S.-based company. The agreement provides an equine product – Equine Gastrafate® - for exclusive distribution in Canada and the U.S. by Bioniche. Equine Gastrafate® is a patented saccharide composition that is used to expedite management of gastrointestinal syndromes characterized by nausea, vomiting, diarrhea, colic and mucosal erosions. This product is often used by equine veterinarians who suspect ulcer colic in horses.
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Trillium Therapeutics
Inc. (Toronto, ON), a privately-held biopharmaceutical company developing proprietary and innovative biologic therapies begin a Phase 1 clinical trial of its experimental drug, TTI-1612, in patients with interstitial cystitis/bladder pain syndrome (IC/BPS). The company has recently received a No Objection Letter to its Clinical Trial Application from Health Canada’s Biologics and Genetic Therapies Directorate. The single ascending dose trial will be conducted at multiple sites across southern Ontario. IC/BPS, also known as Painful Bladder Syndrome, is a chronic, debilitating and poorly treated bladder disease affecting millions of people. The disease is believed to develop as a result of dysfunction in the protective epithelial layer lining the bladder.
Oncolytics Biotech Inc.
(Calgary, AB) has released interim data from its Phase 2 clinical trial using intravenous administration of REOLYSIN® in combination with gemcitabine (Gemzar®) in patients with advanced pancreatic cancer (REO 017) indicating that the clinical study has successfully reached its primary endpoint, and that the drug combination is active. To date, eight patients of 13 evaluable patients in the study had stable disease (SD) for 12 weeks or longer, for a clinical benefit rate (complete response (CR) + partial response (PR) + SD) of 62 per cent. An additional patient had an unconfirmed PR of less than six weeks. The study is using a one sample, two-stage design. In the first stage, 17 patients were to be enrolled, and best response noted. If less than three responses (defined as CR or PR or SD for 12 weeks or more) were observed, the study would have concluded that the combination was inactive and been terminated. If three or more responses were observed among the 17 patents, the study would enroll an additional 16 patients for a total of 33 evaluable patients.
Xenon (Vancouver, BC) announces a strategic alliance with Genentech, a member of the Roche Group, to discover and develop compounds and companion diagnostics for the potential treatment of pain. Under the terms of the agreement, Genentech has an exclusive license to compounds and a non-exclusive license to diagnostics from Xenon for development and commercialization of products. Xenon will receive an undisclosed upfront payment, research funding and is eligible to receive research, development and commercialization milestone payments, totaling up to $646 million for multiple products and indications. In addition, Xenon will receive royalties on sales of products resulting from the collaboration.
Aeterna Zentaris Inc.
(Québec, QC) announces that the U.S. Food and Drug Administration (FDA) has granted Jose M. Garcia, MD, PhD an Investigational New Drug (IND) approval for the initiation of a Phase 2A trial to assess the safety and efficacy of repeated doses of the company’s ghrelin agonist, AEZS-130 (macimorelin), in patients with cancer cachexia. The study is a double-blind, randomized, placebo-controlled Phase 2A trial to test the effects of different doses of the ghrelin agonist, AEZS-130, in 18 to 26 patients with cancercachexia. The study will be conducted under a cooperative research and development agreement (CRADA) with the Michael I. DeBakey Veterans Administration Medical Center which will be funding the study. AEZS-130 will be provided by Aeterna Zentaris.
Allon Therapeutics
Inc. (Vancouver, BC) has been granted a U.S. patent covering the use of Allon’s drug candidates, including its lead product davunetide, and other pipeline products for the treatment of laserinduced retinal damage. This new patent strengthens Allon’s intellectual property estate, which includes 15 patent families, 60 issued patents and over 30 pending applications worldwide. This patent relates to findings recently published in Acta Ophthalmologica which reported that davunetide has neuroprotective effects in an animal model of retinal laser injury. The results suggest the potential of davunetide as a treatment for retinal damage following retinal laser photocoagulation or other types of ophthalmic laser surgeries.
Helix BioPharma Corp.
(Toronto, ON) announces that the “clinical hold” on its investigational new drug application for its Topical Interferon Alpha-2b, Phase 2/3, low-grade cervical lesion efficacy trial has been removed by the U.S. Food and Drug Administration (FDA). The proposed Phase 2/3 trial is planned to be a randomized, double-blind, vehicle-controlled study in patients with cervical intraepithelial neoplasia grade 1 or 2 lesions (CIN 1 or CIN 2 respectively). The intended sample size is 492 female subjects to be randomized in a 2:1 ratio of active to control. Eligible women will be premenopausal subjects aged 18 - 55 years at screening, with histologicallyconfirmed CIN 1 or CIN 2 on colposcopic directed biopsy at screening and high risk human papillomavirus (HPV) infection upon the Hybrid Capture® 2 HPV-DNA test. The proposed primary study endpoint will be the resolution of CIN 1 or CIN 2 at month 12, determined by colposcopic directed cervical biopsy together with Pap smear cytology free of ASC-H (atypical squamous cells that cannot exclude high-grade squamous status), AGUS (atypical glandular cells of undetermined significance), LSIL (low-grade squamous intraepithelial lesions), HSIL (high-grade intraepithelial lesions), and adenocarcinoma in situ (AIS) or adenocarcinoma. The study is designed with a 12-month overall duration per patient, including treatment and follow-up. Helix plans that the results of its U.S. Phase 2/3 trial and the results of its European Phase 3 trial, if successful, will be submitted together in order to seek U.S. and European marketing authorizations for the product for this indication.
FEATURE
BY DR. GUERMAN PASMANIK
Introduction
Sensors for the discovery and identification of both simplest inorganic molecules and organic compounds (including macro- and bio-molecules in living organisms) are mainly used in pharmacology, bio-medicine and chemistry. In general, the progress in sensor technology is determined by our ability to decompose different molecules contained in a “big” volume over elementary “cubicles”, containing identical molecules with equal mass, mobility, optical spectrum, etc. Thus, in particular we are able to determine the presence of low concentration of analytes given the information about processes, say, in human cells.
The most advanced current method of sensing is associated with mass spectrometry. This method has become established as the primary means for protein identification from complex mixtures of biological origin, uncovering the set of proteins that make up human cells. Moreover, due to new approaches based on MALDI imaging, the mapping of protein distribution inside of cells becomes routine procedure. Prospectively measuring the dynamics of map variations make predicting a living cells lifetime achievable. However, this technique is rather expensive and can’t be applied for very big molecules (with sizes of tens and hundreds nanometers). As an alternative, Ion mobility spectrometers allow classification of very big molecules, but to use this method one usually has to know in advance what kind of analytes need to be found. Laser induced fluorescence gives detailed information about analyte spectra, but strong background emission usually masks the low concentrated molecules of interest. Raman spectroscopy can also provide very useful information about molecular structure and outlines features of molecules with close chemical performances, but it too is masked by laser induced fluorescence which usually exceeds Raman emissions on several orders.
As such, there is a need for new methods in detecting molecular structure, and for sensing inorganic molecules and organic compounds. This article proposes a new electrically-controlled (active) filter for the trapping of molecules with high polarizabilities. This new method of molecular sensing (active micro/nano-structured spectroscopy - AMOS) is based on our proprietary technology. The important features of AMOS are its ability to trap bio-molecules based on their polarizability. According to our estimation it is feasible achieving of sensitivity for detection of admixed molecules up better than part per trillion. Likewise, this filter can be a core element of ultra-sensitive analytical spectrometers, filtration of organic compounds in pharmacology, water extraction from air and oil, etc.
Active micro- and nano-structured optical spectrometry (AMOS)
General description of AMOS, technologies to be used and advantages of ps-UV pulses
Among the different methods of sensing, there are separately staying adsorption methods. These methods are characterized by the physical adherence or bonding of ions and molecules onto the surface of another phase. The typical example is a solgel plate, which absorbs water from air. Absorbed material is analyzed, for example, following ionization (in IMS technology) or optical spectra analysis (Laser Induced Fluorescence, Raman or molecular spectroscopy). However the absorber used for molecules of interest accumulation usually is passive.
Recently we have developed a new method of sensing based on a lasermade active filter consisting of dense packaging of micro-holes in dielectric plate matches to vias in structured metal or Indium Tin Oxide electrodes (see Figure 1). By controlling voltage on these electrodes and temperature of the filter it is possible to accumulate molecules and organic compounds near (or inside) of the holes. The physical reason for such accumulation is that the electrostriction interaction of neutral molecules and organic compounds occurs with a high voltage electrical field having a strong non-uniform distribution near and inside of the holes. Especially if such holes are blind (not drilled through a dielectric plate) the bottom of the holes has quasi-periodical spikes (like “stalagmites”) with a typical distance between them achieving up to λ/3, where λ is the laser wavelength in the UV band. For 213 nm, that distance can achieve 71 nm and the transverse size of corresponding spikes is two to three times less (about 25 nm).
Due to high gradient of electrical field (sharp spatial change with nanometer scale) the electrostriction forces attract neutral molecules and organic compounds (in particular proteins) inside of the holes to allow bonding them around spikes. As more polarizability α has molecule as stronger interaction occurs. If the molecule velocity induced by electrostriction force (~ τα E2/m) is comparable to its thermal velocity (~ kT/m), then a molecules bonding becomes strong enough to fix that molecules to the filter. Here we use denotations: k is the Boltzmann constant, T is the filter temperature, τ is the time between molecules collisions (free running time), m is the molecule mass. However, if polarizability α is not very high the molecules can’t be fixed on the filter surface or inside of the hole.
From the above, one will notice that the selective attraction of analyte to the filter’s holes facilitates optical spectral analysis with relatively low background emission. Thus, one of the important features of AMOS is its ability to trap the bio-molecules based on their polarizabilities, and furthermore the larger the polarizability of the molecule, the better the trapping performances of the AMOS as an analytical device.
One important thing to note is that the number of big bio-molecules trapped into each micro-hole (spot) is discrete; correspondingly the molecular performances in individual spots, say, their dipole moments or scattering cross-sections, are also discrete (proportional to the number of molecules). If in the results obtained
Figure 1 Electrically controllable (active) filter
This is a dielectric plate (100 um silica or lavsan) with 2 orthogonal metal (or Indium Tin Oxide) stripes (marked by yellow color) with 50-100 lines/mm deposited on the opposite sides of the plate and having tiny (5-10 um) holes drilled between the stripes.
Set of electrical circuits to excite magnetic field attracting atoms with high magnetic polarizability
there are non-discrete numbers of molecules, then this means that there are some unusual features of trapped molecules. Thus, analyzing all spots (pixels), say, via measuring of response on electrical field scanned over filter cross-section or spectrum of scattered light, we will be able to obtain additional information about the properties of the analyte molecules. Definitely in the case when there is a relatively low concentration of those molecules, vast majority of micro-holes (pixels) don’t have analyte molecules. Nevertheless there may be present spots with analyte molecules. In other words, if analyte molecules are available in any solution, they will also be trapped in certain micro-holes, which make their recognition easier.
The most suitable instrument to execute the investigation of analyte molecules in these considered conditions is Raman scattering of the laser light.
Raman lines unlike fluorescence will provide more information about the geometrical shape of the molecule. In particular, the organic compounds containing the same elementary groups of molecules, but orientated in different directions with respect to the rest of molecules, have different Raman vibration and rotational modes. Recognition of these modes allows for the discovery of organic compounds containing certain defects, for example, a minor group of molecules originated from a parent bio-molecule, but bonded to a molecular body in an unusual manner (from the side or from another end). The processing of obtained results may uncover details of reproductive living organisms including cancer and other diseases at earlier stages.
The use of molecular scattering, in particular so-called Rayleigh wing scattering where the broadening of frequency spectrum and polarization features of scattered light characterize the molecules, is extremely important. Also, we have mentioned the possibility of using surface enhanced Raman scattering (for some molecule complex having a conduction band). And finally regarding Coherent Anti-Stokes Raman Scattering, this method may provide a very high sensitivity especially if we know in advance what kind of analyte is expected. According to our estimation, this means achieving the sensitivity of about one part per trillion.
Figure 2
Technology of AMOS fabrication
on UV picoseconds lasers where the lasers are used in two technology processes: 1) Controllable hole drilling in metal and dielectrics with an accuracy of ~0.1 μm. 2) Controllable removal of thin metal layers deposited on dielectric substrates.
Using these two technology processes we are able to deposit a thin metal film on a fused silica surface and drill 5 to 10 μm holes though that film at a depth of about 10100 μm. In the first technology process, due to its relatively high aspect ratio (the depth of the hole to its diameter) the short wavelength UV laser beam (λ = 266 nm or 213 nm) touches the walls of the hole and is reflected towards the hole bottom initiating light-plasma interaction causing the spatial instability of material removal process. The result of that instability is the development of a pattern with a period of about λ/2n (here n is reflective index of dielectric substrate).
In the second technology process, a high energy UV laser beam focused by cylindrical optics ablates the surface of the substrate with a thin metal film deposited on one of its faces. The period of patterning is about 5 - 1 20 μm. From another face of the same plate the patterning is to be made with the stripes having orthogonal orientation. Then the UV laser beam drills holes between the stripes in each spot.
The voltage given to the structured metal film provides an electrical field up to several kV/cm. That voltage can be variable from spot to spot allowing variable electrical field over the surface. Thus, one can control conditions and create traps for molecules that have different polarizabilities.
Features of AMOS and ways to the improve AMOS design
The performance of the AMOS depends on diameter and surface density of the holes, their depth, the ability of making spikes on the bottom, type of dielectric material and the polarization of the laser beam making the holes. We can separate several types of AMOS technology: 1. Simplified version of
AMOS based on metal-dielectric-metal “sandwich”.
In that case the holes through metal and dielectric are made from opposite sandwich faces. The holes from one face are blind, and holes from opposite side are drilled only through metal. It allows collecting analytes on one side and provides optical spectral analysis on the other; 2. A Structured version of
AMOS based on the ability to control voltage inside of individual holes; 3. A variation of the metaldielectric-metal “sandwich” with simpler (no blind) holes. The main goal of such a device is filtering molecules with high polarizability and ensuring their separation in a gaseous or liquid mixture from other molecules.
FEATURE
One new and interesting structures made by the UV laser ablation is the set micro circuits around the vias or blind holes (see Figure 2)
The electrical current passes these micro-circuits with a typical diameter of 10-20 μm excites the magnetic field inside of the hole. This creates a field that selectively attracts atoms, molecular complex and micro-particles with high magnetic polarizability. This method can open up new possibilities for analysis of metal micro-particles in oil or cleaning oil from such micro-particles, by trapping of atoms of less-common metals, for isotope separation, etc. One could call it “Ω-technology” because each micro-circuit looks like the Ancient Greek letter Ω.
Expected applications and market prospects for AMOS
There are several important applications associated with the use of active filters including the filtering of analytes which would prove useful to the bio-medical analysis, pharmaceutical industry. The filters can also be adapted for ion mobility spectrometry, in separating selected molecules or organic compounds such as those required for the growing of crystals including organic ones. The filters can assist in water extraction from oil to provide improved performance of gasoline or kerosene. Likewise they can be used for high efficient water extraction from air or alternatively air extraction from water.
Author thanks Dr. Sabatino Nacson, Adolf Kleiner and Mrs. Maria Konchalina for fruitful discussion and help.
Dr. Guerman Pasmanik is the lead researcher for Amos Photonics Research Technology, a small technology company, developing and selling optical equipment globally.
Learn more about Separation Solutions on our Lab News Web Portal at
www.bioscienceworld.ca
FEATURE
BY: WILLIAM FAULKNER, KIMBERLEY PHIPPS, THERMO FISHER SCIENTIFIC, RUNCORN, CHESHIRE, UK
Determination of Rosuvastatin
in Human Plasma by SPE-LC-MS/MS using SOLA and Accucore RP-MS column
A SIMPLE, RAPID AND SENSITIVE PROCEDURE for the determination of rosuvastatin in human plasma by liquid chromatography-tandem mass spectrometry was developed and evaluated. The drug was isolated from within the plasma matrix using a Thermo Scientific SOLA 96 well plate, and the components of the resultant extracts were separated on a Thermo Scientific Accucore RP-MS column under reversed-phase, gradient conditions. Detection was performed on a triple quadrupole mass spectrometer under positive polarity, heated electrospray ionisation (HESI) conditions operating in selected reaction monitoring (SRM) mode.
The analytical procedure is both accurate and precise, and is characterized by high levels of recovery and an absence of any significant matrix interfering effects.
Introduction
Rosuvastatin [(3R, 5S, 6E)-7 - [4- (4 - fluorophenyl) -2- (N-methylmethanesulphonamido) -6- (propan-2-yl) pyrimidin-5-yl] -3, 5 - dihydroxyhept -6-enoic acid] is a synthetic, orally administered member of the ‘statin’ class of cholesterol lowering drugs. This particular statin is marketed by Astra Zeneca as ‘Crestor’. Employed as an adjunct to dietary modification, the drug is used to treat primary hypercholesterolaemia, mixed dyslipidaemia and hypertriglyceridaemia in an attempt to reduce the risk of atherosclerosis and poor cardiovascular health.
In terms of the mechanism of its action, rosuvastatin is a selective and competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. This particular enzyme catalyses the conversion of HMG-CoA to mevalonate, a precursor of cholesterol.
Accucore™ columns are packed with silica particles that have been manufactured using a core enhanced technology. These particles comprise a solid core upon which a porous layer is deposited. This configuration minimizes diffusional (mass transfer) pathways and permits the use of shorter columns and higher flow rates to achieve remarkably fast, highly efficient and highly resolved separations. In contrast to totally porous sub-2 μm particles, columns containing the solid core particles generate a competitive number of theoretical plates, but, with greatly reduced backpressures.
Accucore RP-MS columns employ an alkyl chain of an optimized length in order to provide a more effective coverage of the silica surface. The immobilised phase exhibits a slightly lower retentivity than that offered by traditional C18 phases, but, the resultant high efficiencies and excellent peak symmetries make this the phase of choice for those chromatographic procedures which demand detection by mass spectrometry.
SOLA™ is a revolutionary new solid phase extraction (SPE) product. This first in class SPE product range introduces next-generation, innovative, technological advancements which offer unparalleled performance in comparison to conventional SPE and products intended for the precipitation of proteins and phospholipids. The benefits of using SOLA products include: (1) higher levels of reproducibility, (2) improvements in the cleanliness of extracts, (3) reduction in elution volumes (4) increases in procedural sensitivity.
SOLA products have significant advantages for the analyst when isolating compounds from complex matrices, particularly in high throughput bioanalytical and clinical laboratories where higher speed of analysis and the ability to use smaller quantities of sample and solvent are critical. The increased performance of SOLA products gives greater confidence in the accuracy of analytical results and lowers cost without compromising ease of use or requiring complex method development.
A number of researchers have reported the measurement of rosuvastatin in human plasma and pharmaceutical formulations.1,2 Typical chromatographic approaches include separation using C18 phases (via hydrophobic interactions) and retention of the ionized molecule via an ion-exchange mechanism.
The purpose of this particular study is to demonstrate the effectiveness of a combination of SOLA products (reversed phase) solid phase extraction material and an Accucore RP-MS column for the determination of rosuvastatin in human plasma by liquid chromatography tandem mass spectrometry.
Results
Linearity of response
The relationship between analytical response and concentration was investigated over the range 1-1000 ng/ mL.
A calibration line was constructed using data derived from extracted matrix-fortified samples at eight levels of concentration (excluding blanks).
Calibration lines were run at the beginning and end of the analytical sequence with the bottom and top standards being extracted in duplicate in both lines. All data were used to assess the degree of linearity.
A graphical plot of relative response (A /A ) as a function of the concenstd istd tration of rosuvastatin is shown in Figure 1. Calibration data are summarised in Table 1.
The analytical response was found to be linear (using a 1/x weighted regression algorithm) with a coefficient of determination (r2) of 0.9984 in the range 1 - 1000 ng/mL.
Assessment of accuracy and precision
Procedural accuracy and precision were evaluated by replicate (n = 6) examination of extracted QC samples at three levels of concentration. A summary of the results is shown in Table 2.
The accuracy and precision of the analytical procedure were found to fall comfortably within the limits of acceptance generally applied to bioanalytical methods.
Figure 1
2.5
2.0
1.5
1.0 Linearity of response over the dynamic range 1 – 1000 ng/mL r2 = 0.9984 SRM derived from examination of extracted standard S1 (1 ng/mL) SRM derived from examination of an unfortified, extracted (blank) plasma sample
0.5
Figure 2
100
90
80
Relative Abundance 70
60
50
40
30
20
Figure 3
100
90
Relative Abundance 80
70
60
50
40
30
20
Evaluation of recovery
The recovery of analyte was assessed by comparison of the measured concentrations of rosuvastatin in matrix extracted QC samples with those concentrations found in post-extraction spiked samples which had been fortified at the same level (See Table 3).
The level of analyte recovery (99.3 %) and the precision (% RSD = 4.88) between replicates demonstrate that both the efficiency of the extraction procedure and its repeatability are substantially more than satisfactory.
Statistical assessment is based upon data derived from the replicate examination of both pre- (n = 6) and postextracted (n = 3) plasma samples.
Evaluation of matrix effect
The existence of a matrix effect was determined by comparison of the measured concentrations of rosuvastatin in post-extracted fortified plasma samples with those concentrations found in the reference standards that had been fortified at the same level. The results, summarized in Table 4, indicate that there is no significant matrix effect.
Statistical assessment is based upon data derived from the replicate examination of both post-extracted plasma samples (n = 3) and reference standards (n = 6).
Specificity and sensitivity
SRM chromatograms derived from the examination of the unfortified and fortified plasma samples are shown in Figures 2 and 3.
It is evident that the unfortified plasma sample contains an impurity (Tr = 1.92 minutes). However, under the adopted chromatographic conditions, the separation is sufficient to prevent any overlap of the response from this endogenous plasma species upon the principal analytical response (Tr = 1.49 minutes).
The SRM derived from the zero blank is shown in Figure 4. The mass spectral evidence suggests that there is no proton exchange and concomitant conversion of the deuterated to the non-deuterated form.
Conclusion
An analytical procedure based upon SPE-LC-MS/MS for the determination of rosuvastatin in human plasma was successfully developed and evaluated. The procedure was found to exhibit good linearity (r2 = 0.9984) for concentrations of rosuvastatin in the range 1 - 1000 ng/mL. The accuracy and precision for all samples examined were found to be < 6.1 % and < 5.8 % respectively. At a concentration of 400 ng/mL, the level of recovery of analyte (99.3 %) and its repeatability (% RSD = 4.9 %) were both found to be excellent. There was no significant matrix effect.
The performance characteristics of the method combined with its simplicity and rapidity mean that it can be adopted routinely in clinical environments.
References
1. Pelat, M.; Dessy, C.; Massion, P.;
Desager, J.-P.; Feron, O.; Balligand, J.-L., J. Circ. 2003, 107, 2480 2. Trivedi, R.K.; Kallem, R.R.; Mullangi, R.; Srinivas, N.R., J. Of
Pharm. Biomed. Anal. 2005, 39(3-4), 661-669
Experimental Details
Chemicals and Reagents
Fisher Scientific Water (LC-MS grade)
Part Number
W/0112/17 Fisher Scientific Methanol (LC-MS grade) M/4062/17 Fisher Scientific Formic acid (‘Optima’, 90 %, LC-MS grade) A117-50 Fisher Scientific Propan-2-ol (HPLC grade, 99.5+ %) P/7507/17 Fisher Scientific Acetonitrile (LC-MS grade) A/0638/17 Fisher Scientific Acetone (AR grade, 99.8+ %) A/0600/15 Human plasma (Supplied by Seralab, lithium heparin as anticoagulant)
Sample Handling Equipment
Fisher Scientific Finnpipette F2 pipettor kit
Part Number PMP-020-220F
10 μL - 100 μL, 100 μL - 1000 μL, 1 mL – 10 mL Fisher Scientific Finntip pipette tips, 10 μL Fisher Scientific Finntip pipette tips, 200 μL Fisher Scientific Finntip pipette tips, 1000 μL Fisher Scientific Finntip pipette tips, 10 mL
PMP-107-110W PMP-107-600F PMP-103-206K PMP-107-040R Thermo Scientific borosilicate glass vials 60180-600 (2 mL, 12 mm x 32 mm) with 8 mm black screw cap fitted with a silicone/PTFE seal
Solid Phase Extraction Hardware
Thermo Scientific positive pressure SPE manifold (capable of processing 96-well microplates). Solvent evaporation system (capable of processing 96-well microplates).
SPE Apparatus Part Number
SOLA 96-well plate (10 mg/2 mL)
FEATURE
60309-001
Calibration Standards
A primary standard of rosuvastatin was prepared in 1:4 (v/v) MeOH-water at a concentration of 105 ng/mL. Secondary standards (SS1 – SS8) were prepared by subsequent serial dilution of the primary standard in 1:4 (v/v) MeOH-water.
4.5x10-LabFocus:2 2011/08/09 2:46 PM Page 1
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FEATURE
A primary standard of d6-rosuvastatin, to be used as internal standard, was prepared in 1:4 (v/v) MeOH/water at a concentration of 5000 ng/mL. Calibration standards of rosuvastatin were prepared at eight different levels of concentration (i.e., 1, 2, 10, 50, 250, 500, 900 and 1000 ng/mL) by fortification of the plasma matrix (100 μL) with a measured quantity (10 μL) of appropriate stock standard. The internal standard (d6-rosuvastatin) was added at the 500 ng/mL level into each of the calibrants. Standards S1 and S8 were prepared in duplicate whilst the remaining standards (S2 – S7) were prepared in singlet
Quality Control (QC) Standards
QC standards of rosuvastatin were prepared in replicate (n = 6) at three levels of concentration (i.e., 3, 400 and 750 ng/mL) by fortification of the plasma matrix (100 μL) with a measured quantity (10 μL) of appropriate spiking solution. The internal standard (d6-rosuvastatin) was added at 500 ng/mL.
QC Standards for Post-Extraction Fortification (i.e., ‘overspiking’)
Unfortified plasma samples (n = 9) were extracted in triplicate and fortified post extraction at three levels of concentration, i.e., 1, 400 and 750 ng/mL.
Reference (unextracted) Standards
Reference standards (which were not subject to extraction) were prepared in replicate (n=6) at three different levels of concentration by the introduction of a measured quantity (10 μl) of appropriate spiking solution ([Ros.]QCLLOQ = 10, [Ros.]QCMED = 4000, [Ros.]QCHIGH = 7500 ng/mL) and internal standard (d6 rosuvastatin, 10 μL, [d6-Ros.] = 104 ng/mL) into 1:4 (v/v) MeOH-water (180 μL).
Blank Samples
Double blank samples were prepared in duplicate by the introduction of water (20 μL) into plasma (100 μL). These were unfortified with respect to both rosuvastatin and its deuterated analogue.
Zero Blank Sample
The ‘zero blank’ comprised plasma (100 μL) spiked with internal standard only.
Isolation of Rosuvastatin using SOLA (Extraction Procedure)
The extraction was carried out using a positive pressure SPE manifold capable of processing 96-well microplates. • Condition SOLA with MeOH (1.0 mL at 1.0 mL/minute) • Condition SOLA with water (1.0 mL at 1.0 mL/minute) • Load sample (100 μL) and allow to permeate SOLA material under gravity • Wash with 0.1 % (v/v) formic acid (500 μL at 1.0 mL/minute) • Wash with 10 % (v/v) MeOH (500 μL at 1.0 mL/minute) • Elute with 90 % (v/v) MeOH (2 x 200 μL at 1.0 mL/minute) The methanolic extracts were evaporated to dryness under a stream of nitrogen at 40 °C and reconstituted in 1:4 (v/v) MeOH-water (200 μL).
Instrumentation
Separation was carried out using a Thermo Scientific Accela 600 pump interfaced to both a Thermo Scientific Accela Open Autosampler, and, a Thermo Scientific TSQ Vantage triple stage quadrupole mass spectrometer.
Chromatographic Conditions Part Number
Column: Thermo Scientific Accucore RP-MS 17626-052130 2.6 μm, 50 x 2.1 mm Guard column: Accucore Defender (RP-MS) 17626-012105 guard column 2.6 μm, 10 x 2.1 mm Uniguard direct connection guard cartridge holder (2.1 mm I.D) Mobile phase: (A) Water + formic acid at 0.1 % (v/v)
(B) MeOH + formic acid at 0.1 % (v/v) 852-00
Gradient: Time (min) 0 0.1 % B 5 5
1.0 1.60 1.62 3.60
95 95 5 5 Flow rate: 0.75 mL/min Column temperature: 60 °C Detection: MS Injection volume: 15 μL Syringe volume: 100 μL Syringe flush: Strong solvent - 9:9:2 (v/v) MeCN/iPrOH/acetone Weak solvent - 1:4 (v/v) MeCN-water Autosampler temperature - 10 °C Run time: 3.60 minutes
Mass spectrometric conditions
Ionisation parameters: Heated electrospray operating in positive polarity mode (HESI-2 probe) Spray voltage: 3000 V Vaporiser temperature: 475 °C
Sheath gas pressure: Ion sweep gas pressure: Capillary temperature: Declustering voltage: Collision pressure: 65 0.5 300 °C 0 V 1.5 mTorr
MS acquisition parameters:
Quantification was performed by selected reaction monitoring (SRM) using the precursor-to-product combinations shown below:
Scan type: Peak width: SRM Q1 - 0.7 (FWHM) Q3 - 0.7 (FWHM)
Scan width: 0.2 m/z
Scan time: Divert valve:
MS acquisition time: 3.60 minutes
Data Processing
All data were processed using Thermo Scientific LCQuan (v. 2.6) software. Algorithm for integration - ICIS
FEATURE
Figure 4
SRM derived from examination of the zero blank (upper trace: d6-rosuvastatin, lower trace: undeuterated analogue
100
90
80
Relative Abundance 70
60
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40
30
20
10
0
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Time (min)
100
90
80
Relative Abundance 70
60
50
40
30
20
10
0
0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Time (min)
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