Biomedical subjects
C A James
Publications and source records attributed to C A James.
Bioanalytical method validation: a risk-based approach?
Bioanalysis frequently involves the measurement of very low analyte concentrations in complex and potentially variable matrices. It is not possible to test in validation every possible circumstance that may be encountered when analyzing study samples; logically, therefore, some risk of obtaining erroneous results exists when validated methods are applied to study samples. An initial attempt has been made to apply a risk management tool to the bioanalytical situation, with the hope that this will stimulate further discussion on the idea of more formally addressing "risk" with regards to bioanalytical method validation.
Quantitation of SU1 1248, an oral multi-target tyrosine kinase inhibitor, and its metabolite in monkey tissues by liquid chromatograph with tandem mass spectrometry following semi-automated liquid-liquid extraction.
SU11248 is a potent inhibitor of PDGFR, VEGFR, KIT, and Flt3, and is currently under Phase I clinical evaluation as an anticancer drug. A sensitive and specific analytical method for the quantitation of SU11248 and its metabolite in several monkey tissues (liver, kidney, brain and white fat) using LC-MS-MS following semi-automated liquid-liquid extraction (LLE) was developed and validated. Amounts of 50 mg of tissue were homogenized using an ultrasonic processor. After addition of the stable labelled internal standard (IS) and ammonium hydroxide (0.3%), samples were extracted with 2.5 ml of tert-butyl methyl ether. Following centrifugation, aliquots of 1.8 ml of the organic phase were transferred into a 96-well plate. The Packard Multiprobe II robotic liquid handler was used to perform all steps mentioned above. The organic phase was dried and the residue was reconstituted with 800 microl of 15 mM ammonium formate buffer solution (pH 3.25) using a Tomtec Quadra 96 workstation. Aliquots of 10 microl of the resulting solution were injected into the LC-MS-MS system. A Symmetry Shield C8 column (50 mm x 2.1 mm, 3.5 microm) was used to perform the chromatographic analysis. The mobile phase was 15 mM ammonium formate buffer solution (pH 3.25)-acetonitrile (74:26 (v/v)) with a flow-rate of 0.35 ml/min. Retention times of the metabolite and SU11248 were about 2.5 and 3.5 min, respectively. Total cycle time was 5 min. MS detection used the Applied Biosystems-MDS Sciex API 3000 with TurbolonSpray interface and multiple reaction monitoring (MRM) operated in positive ion mode. The method was validated for both compounds over the calibration range of about 2 and 2000 ng/g. The suitability and robustness of the method for in vivo samples were confirmed by analysis of monkey tissues from animals dosed with SU11248.
Simultaneous determination of estramustine phosphate and its four metabolites in human plasma by liquid chromatography-ionspray mass spectrometry.
A sensitive and selective method, using liquid chromatography-ionspray mass spectrometry, was developed and validated for the simultaneous determination of Estracyt (estramustine phosphate) and its four metabolites, estramustine, estromustine, estrone and estradiol, in human plasma. Deuterated internal standards were available for all analytes. The five compounds were extracted from plasma by protein precipitation with acetonitrile. The chromatographic separation was performed using a Zorbax SB C18, (150 x 4.6 mm i.d., 5 microm) reversed-phase column under gradient conditions with a mobile phase containing 2 mm ammonium acetate buffer (pH 6.8) and acetonitrile. MS detection was by electrospray ionization with multiple reaction monitoring in the positive ion mode for estramustine phosphate, estromustine and estramustine, and in the negative ion mode for estrone and estradiol. The limit of quantitation was 10 ng/mL for estramustine phosphate, 3 ng/mL for estromustine, estramustine and estrone and 30 ng/mL for estradiol. Linearity was verified from these LLOQs up to about 4000 ng/mL for the parent drug and 2000 ng/mL for the metabolites. Inter-day precision and accuracy values were all less than 15%. This assay was applied successfully to the routine analysis of human plasma samples collected in cancer patients administered estramustine phosphate intravenously.
Simultaneous determination of JTT-501 and its main metabolite in human plasma by liquid chromatography-ionspray mass spectrometry.
An LC-MS-MS analytical method was developed for the determination of a new antidiabetic agent, JTT-501 and its main metabolite (JTP-20604) in human plasma. The compounds were isolated from plasma by protein precipitation before analysis by HPLC with atmospheric pressure positive ionisation MS-MS detection. An isotopically labelled analog of JTT-501 was used as the internal standard. Linearity was demonstrated over the calibration range of about 5-10000 ng/ml for both compounds. The assay was validated with respect to accuracy, precision and analyte stability. This method was used for the determination of plasma concentrations for the two compounds in a clinical tolerability study. A cross-validation exercise between two different mass spectrometers, used for the determination of clinical samples, is also reported.
Quantitative determination of paclitaxel in human plasma using semi-automated liquid-liquid extraction in conjunction with liquid chromatography/tandem mass spectrometry.
This paper describes a high-throughput sample preparation procedure combined with LC-MS/MS analysis to measure paclitaxel in human plasma. Paclitaxel and an internal standard were extracted from plasma by a semi-automated robotic method using liquid-liquid extraction. Thereafter compounds were separated on a RP C18 column. Detection was by a PE Sciex API 3000 mass spectrometer equipped with a TurboIonSpray interface. The compounds were detected in positive ion mode using the mass transition m/z 854.6-->286.2 and m/z 831.6-->263.2 for paclitaxel and the internal standard, respectively. The limit of quantitation for paclitaxel was 1 ng/ml with an imprecision of 5.2% following extraction of 0.1 ml of plasma. Linearity was confirmed over the whole calibration range (1-1000 ng/ml) with correlation coefficients higher than 0.99 indicating good fits of the regression models. The inter and intra-day precision was better than 9.5% and the accuracy ranged from 90.3 to 104.4%. The assay was simple, fast, specific and exhibited excellent ruggedness.
Determination of PNU-248686A, a novel matrix metalloproteinase inhibitor, in human plasma by liquid chromatography-tandem mass spectrometry, following protein precipitation in the 96-well plate format.
A sensitive, specific and high-throughput analytical method for the quantitation of PNU-248686A (I), in human plasma has been developed. I, sodium (2R)-3-[[(4'-chloro(1,1'-biphenyl)-4-yl]sulfonyl]-2-hydroxy-2-[(phenylsulfanyl)methyl] propanoate, is an orally active matrix metalloproteinase (MMP) inhibitor developed for the treatment of solid tumors over-expressing MMPs. Concentrations of I, as free acid, were determined in human plasma by LC-MS-MS after plasma protein precipitation in the 96-well plate format. Aliquots of plasma (50 microl) were placed into the plates and 0.2 ml of methanol was added. The plates were shaken for 5 min and centrifuged at 1500 g for 10 min. Aliquots of 10 microl of the supernatants were then directly injected into the LC-MS-MS system. A Symmetry Shield C. column (50 x 2.1 mm, 3.5 microm) was used to perform the chromatographic analysis. The mobile phase was 5 mM ammonium formate buffer solution pH 5.0-acetonitrile (60:40. v/v) with a flow-rate of 0.3 ml/min. Retention time of I was about 1.2 min. Total cycle time was 2.5 min. MS detection used the Applied Biosystems-MDS Sciex API 3000 with TurbolonSpray interface and single reaction monitoring (461 --> 251 m/z transition) operated in negative ion mode. Calibration curves were constructed by plotting the area of the compound (y) against its concentration (x). A weighed linear regression (weighting factor 1/x(2)) was used to calculate I concentrations in quality control and unknown samples. The method was fully validated over the range of 5.0-5000 ng/ml. The suitability and robustness of the method for in vivo samples was confirmed by analysis of plasma samples from a pilot clinical study.
LC-MS-MS determination of nemorubicin (methoxymorpholinyldoxorubicin, PNU-152243A) and its 13-OH metabolite (PNU-155051A) in human plasma.
A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for quantitative determination of nemorubicin, (PNU-152243A, 3'-deamino-3'[2(S)-methoxy-4-morpholinyl]doxorubicin) hydrocloride and its reduced metabolite PNU-155051 in human plasma has been developed and validated. The method involved solid phase extraction (SPE) in 96-well plates. Plasma samples (0.5 ml plasma, spiked with doxorubicin as internal standard and diluted with 0.5 ml of 0.01 M borate buffer, pH 8.4) were extracted using Oasis HLB SPE material. The elution of PNU-152243, PNU-155051 and of IS was performed with 1 ml of methanol:0.1 M formic acid mixture (90:10, v/v). The organic phase was reduced to dryness under a stream of nitrogen at 20 degrees C and the residue was reconstituted with 0.25 ml of 10 mM ammonium formate buffer pH 4.15:acetonitrile mixture (90:10, v/v). Aliquots of 60 microl of the resulting solution were injected onto the LC-MS-MS system. A Zorbax SB C18 column (2.1 x 150 mm, 3.5 microm) was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 10 mM pH 4.15:acetonitrile (73:27, v/v) with a flow-rate of 0.2 ml/min. Detection was achieved by a PE-SCIEX API 3000 with Turbo IonSpray interface, and multiple reaction monitoring (645 --> 321 for PNU-152243, 647 --> 363 for PNU-155051 and 545 --> 345 m/z for doxorubicin) operated in positive ion mode. A weighted linear regression was used to calculate PNU-152243 and PNU-155051 concentrations in QC and unknown samples. Linearity, precision, accuracy and recovery of the method were evaluated over the concentration range of 0.1-5 ng/ml for both compounds. No interference from blank human plasma was observed. The suitability of the method for in vivo samples was assessed by the analysis of samples obtained from patients who had received a single intrahepatic artery dose of PNU-152243A.
Congenital nonprogressive hemangioma: a distinct clinicopathologic entity unlike infantile hemangioma.
BACKGROUND: Infantile hemangiomas are common tumors, distinctive for their perinatal presentation, rapid growth during the first year of life, and subsequent involution-and for their expression of a unique immunophenotype shared by placental microvessels. Occasional "hemangiomas" differ from the classic form in presenting fully formed at birth, then following a static or rapidly involuting course. These congenitally fully developed lesions have generally been assumed to be clinical variants of more typical, postnatally developing hemangiomas. This assumption has not been tested by rigorous histologic and immunophenotypic comparisons. OBJECTIVE: To compare the histologic and immunohistochemical features of congenital nonprogressive hemangiomas with those of typical, postnatally proliferating, hemangiomas. DESIGN: All cellular vascular tumors resected from infants younger than 4 months at Arkansas Children's Hospital, Little Rock, over the past 20 years (43 lesions from 36 patients) were first characterized histologically and immunohistochemically, then clinically by chart review. SETTING: A university-affiliated pediatric hospital. MAIN OUTCOME MEASURES: Histologic appearance, immunoreactivity for the infantile hemangioma-associated antigens GLUT1 and LeY, and clinical behavior. RESULTS: Congenital nonprogressive hemangiomas differed from postnatally proliferating infantile hemangiomas in histologic appearance and immunohistochemical profile. Distinguishing pathologic features of these tumors were lobules of capillaries set within densely fibrotic stroma containing hemosiderin deposits; focal lobular thrombosis and sclerosis; frequent association with multiple thin-walled vessels; absence of "intermingling" of the neovasculature with normal tissue elements; and lack of immunoreactivity for GLUT1 and LeY. CONCLUSION: Congenital nonprogressive hemangiomas are histologically and immunophenotypically distinct from classically presenting hemangiomas of infancy, unlikely to be related to the latter in pathogenesis.
Congenital cerebral and cerebrovascular anomalies: magnetic resonance imaging.
Magnetic resonance is the imaging modality of choice for the evaluation of infants and children with known or suspected cerebral and cerebrovascular abnormalities. Entities described include cephaloceles, holoprosencephaly, dysgenesis of corpus callosum, and anomalies of neuronal migration. Congenital vascular lesions described include aneurysm, arteriovenous malformation, cavernous malformation, and Galenic malformations.
Ophthalmologic disease in children.
MR imaging has become essential in the evaluation of a spectrum of ophthalmologic diseases in children. Orbit, globe, optic nerve, and optic tract disease processes that are frequently evaluated by MR imaging include congenital malformations, and inflammatory, neoplastic, and traumatic lesions. MR imaging evaluation aids in differentiating these lesions for diagnosis, the extent of disease, treatment planning, follow-up, and prognosis.
Determination of 4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyldaunorubicin+ ++ and its 13-hydroxy metabolite by direct injection of human plasma into a column-switching liquid chromatography system with mass spectrometric detection.
A selective, sensitive and fully automated column-switching LC system using direct injection of human plasma followed by mass spectrometry (MS) detection was developed and validated to determine the concentrations of 4-demethoxy-3'-deamino-3'-aziridinyl-4'-methylsulphonyldaunorubicin++ + (PNU-159548) and its 13-hydroxy metabolite (PNU-169884). A 50-microl human plasma sample was directly introduced into a C4-alkyl-diol silica clean-up column separating analytes from proteins and polar endogenous compounds using water and methanol as the mobile phase. The fraction containing PNU-159548 and its metabolite was back-flushed and transferred to the analytical column. The compounds were separated using a Zorbax SB C8 column (150x4.6 mm, 5 microm) under gradient conditions with the mobile phase containing acetonitrile and 2 mM ammonium formate, pH 3.5. MS detection was by atmospheric pressure ionisation with multiple reaction monitoring in positive ion mode. Linearity was demonstrated over the calibration range of 0.051-10.291 ng/ml for PNU-159548 and 0.104-10.434 ng/ml for PNU-169884. The assay was validated with respect to accuracy, precision and analyte stability. On the basis of the validation data, the developed analytical method was found to be suitable for use in Phase I clinical studies.
Determination of PNU 157706, a new dual inhibitor of 5alpha-reductase, in rat plasma by high-performance liquid chromatography with ultraviolet detection.
A HPLC procedure was developed and validated for determining nanogram per milliliter concentrations of the dual 5alpha-reductase inhibitor PNU 157706 in rat plasma. The compound was extracted from plasma with diethyl ether followed by purification using a CN cartridge. The chromatographic separation was performed with a C18 column using a water-acetonitrile-methanol mixture as eluent. UV detection at 210 nm was used for the quantification of the compound over the concentration range 5-500 ng/ml plasma. The method has a lower limit of quantification of 5 ng/ml and good precision and accuracy. This method has performed well during analysis of several toxicokinetic and pharmacokinetic studies in the rat.
Transcranial Doppler, MRA, and MRI as a screening examination for cerebrovascular disease in patients with sickle cell anemia: an 8-year study.
OBJECTIVE: The authors previously reported five transcranial Doppler ultrasonography (TCD) findings as significant in detecting clinical cerebrovascular disease in a 4-year study in patients with sickle cell disease. This is a follow-up to evaluate the validity of the original findings over another 4-year period during which the study population doubled. A clinical follow-up of the original asymptomatic sickle cell patients with positive TCD, MRA, and MRI was also made. MATERIALS AND METHODS: Over an 8-year period TCD, MRI, and MRA were prospectively performed in 90 sickle cell patients who were clinically asymptomatic for stroke and in 27 sickle cell patients with clinical stroke. RESULTS: Of the 4 out of original 46 control patients in 1992 who had positive MRA and TCD, 3 have subsequently had clinical stroke. None of the 9 original patients with positive TCD and positive MRI but negative MRA have developed stroke. All five original TCD indicators of disease were still significant (P < 0.05) for detecting clinical disease: maximum velocity in ophthalmic artery (OA) > 35 cm/s, mean velocity in middle cerebral artery (MCA) > 170 cm/s, resistive index (RI) in OA < 50, velocity in OA greater than in MCA, and velocity in posterior cerebral (PCA), vertebral, or basilar arteries greater than in MCA. An RI of < 60 in the DA was also now found to be significant [corrected]. Four additional factors were also significant: turbulence, PCA or ACA without MCA, RI < 30, and maximum velocity in MCA > 200 cm/s. CONCLUSION: Positive MRA with a positive TCD in an asymptomatic patient in long-term follow-up suggests a trend for developing clinical stroke. A 4- to 8-year follow-up of nine patients with positive TCD, positive MRI, but not positive MRA did not show development of clinical stroke. Nine Doppler findings are significant in screening for clinically symptomatic vascular disease in sickle cell patients. It is recommended that children with sickle cell disease be screened for cerebrovascular disease with TCD. If one or two indicators of abnormality are present, MRA is recommended. If the MRA is positive, the patient may be considered for transfusion therapy or other treatment for prevention of stroke.
Intracranial aneurysms in infants and children.
BACKGROUND: The diagnosis and imaging of pediatric aneurysms has changed since the advent of MR and MRA. OBJECTIVE: To update the literature on pediatric aneurysms and better define the appropriate work-up of childhood aneurysms in 1997. MATERIALS AND METHODS: Retrospective review of 21 children (12 boys, 9 girls) with 25 aneurysms from three institutions over a 20-year period was performed. Imaging studies were mixed and included CT (19 patients), MR (11 patients), MRA (6 patients) and angiography (18 patients). RESULTS: Eighteen of 25 aneurysms were congenital saccular, 6 were mycotic, and 1 was post-traumatic. Of these, 44 % were in the posterior circulation. Nine aneurysms arose from distal arterial branches. Forty percent were large (between 1-2.5 cm) and 16 % were giant (> 2.5 cm). CT and MR showed hemorrhage, and frequently revealed the aneurysms as a focal mass with or without enhancement and flow void. Six children had MRA which revealed aneurysms in four patients. All patients with MRA had corresponding conventional angiography. CONCLUSION: Characteristics of pediatric aneurysms include diversity of type, increased incidence in the posterior fossa, peripheral location, and large size. CT, MR and MRA are useful in the diagnosis with conventional angiography essential for preoperative planning.
Reconversion of bone marrow in Gaucher disease treated with enzyme therapy documented by MR.
BACKGROUND: Skeletal complications are responsible for significant morbidity in Gaucher patients. Plain radiographs have been unreliable in assessing bone marrow infiltration and activity. A way to assess bone marrow improvement is needed during enzyme therapy. OBJECTIVE: The purpose of this paper is to assess the usefulness of MR in following improvement of abnormal bone marrow in Gaucher patients on enzyme therapy. MATERIALS AND METHODS: Three patients aged 2, 7, and 24 years underwent serial MR scans of the lower extremities before and during treatment with Alglucerase (two patients) and Imiglucerase (one patient). T1-weighted, T2-weighted, STIR and FSE T2-weighted images were utilized. Two patients were imaged after 16 months of therapy, and one patient was imaged after 6 months of therapy. RESULTS: All patients had improvement in marrow signal consistent with partial reconversion to fatty marrow during treatment. The findings were more marked after prolonged therapy. T1-weighted images demonstrated findings most clearly. CONCLUSION: MR consistently showed improvement in marrow signal in Gaucher patients on enzyme therapy. As smaller doses of enzyme therapy are the trend, MR can be utilized to determine if therapy is effecting a change in the bone marrow.
Doxorubicin pharmacokinetics: the effect of abnormal liver biochemistry tests.
We studied variability in doxorubicin pharmacokinetics in 24 patients with abnormal liver biochemistry tests. Blood samples were collected after the first cycle of single-agent doxorubicin given as an i.v. bolus and plasma levels were measured by high-performance liquid chromatography. The relationship between doxorubicin clearance (dose/AUC) and liver biochemistry tests (AST, bilirubin, albumin, alkaline phosphatase and indocyanine green clearance) was investigated. Patients with a raised bilirubin level had reduced doxorubicin clearance, but there was no clear relationship between the extent of this elevation and the reduction in doxorubicin clearance. Doxorubicin clearance was lower in patients with an isolated increase in AST than in those with normal liver biochemistry, but this difference was not statistically significant. Nevertheless, there was a significant correlation between reduced doxorubicin clearance and both raised serum AST levels and low indocyanine green clearance. These pharmacokinetic data suggest that current dose reductions based solely on the extent to which bilirubin is elevated may not be optimal.