Enantiomeric analysis of overlapped chromatographic profiles in the presence of interferences. Determination of ibuprofen in a pharmaceutical formulation containing homatropine.

In this work, we studied the combination of chemometric methods with chromatographic separations as a strategy applied to the analysis of enantiomers when complete enantioseparation is difficult or requires long analysis times and, in addition, the target signals have interference from the matrix. We present the determination of ibuprofen enantiomers in pharmaceutical formulations containing homatropine as interference by chiral HPLC-DAD detection in combination with partial least-squares algorithms. The method has been applied to samples containing enantiomeric ratios from 95:5 to 99.5:0.5 and coelution of interferents. The results were validated using univariate calibration and without homatropine. Relative error of the method was less than 4.0%, for both enantiomers. Limits of detection (LOD) and quantification (LOQ) for (S)-(+)-ibuprofen were 4.96×10-10 and 1.50×10-9mol, respectively. LOD and LOQ for the R-(-)-ibuprofen were LOD=1.60×10-11mol and LOQ=4.85×10-11mol, respectively. Finally, the chemometric method was applied to the determination of enantiomeric purity of commercial pharmaceuticals. The ultimate goal of this research was the development of rapid, reliable, and robust methods for assessing enantiomeric purity by conventional diode array detector assisted by chemometric tools.

Automatized sspKa measurements of dihydrogen phosphate and Tris(hydroxymethyl) aminomethane in acetonitrile/water mixtures from 20 to 60°C.

We measured pKa values of Tris(hydroxymethyl)aminomethane and dihydrogen phosphate; both are commonly used to prepare buffers for reverse-phase liquid chromatography (RPLC), in acetonitrile/water mixtures from 0% to 70% (v/v) (64.6% (w/w)) acetonitrile and at 20, 30, 40, 50, and 60°C. The procedure is based on potentiometric measurements of pH of buffer solutions of variable solvent compositions using a glass electrode and a novel automated system. The method consists in the controlled additions of small volumes of a thermostated solution from an automatic buret into another isothermal solution containing exactly the same buffer-component concentrations, but a different solvent composition. The continuous changes in the solvent composition induce changes in the potentials. Thus, only two sequences of additions are needed: increasing the amount of acetonitrile from pure water and decreasing the content of acetonitrile from 70% (v/v) (64.6% (w/w)). In the procedure with homemade apparatus, times for additions, stirring, homogenization, and data acquisition are entirely controlled by software programmed for this specific routine. This rapid, fully automated method was applied to acquire more than 40 potential data covering the whole composition range (at each temperature) in about two hours and allowed a systematic study of the effect of temperature and acetonitrile composition on acid-base equilibria of two widely used substances to control pH close to 7. The experimental pKa results were fitted to empirical functions between pKa and temperature and acetonitrile composition. These equations allowed predictions of pKa to estimate the pH of mixtures at any composition and temperature, which would be very useful, for instance, during chromatographic method development.

Development of an ionic liquid-based dispersive liquid-liquid microextraction method for the determination of nifurtimox and benznidazole in human plasma.

Dispersive ionic liquid-liquid microextraction combined with liquid chromatography and UV detection was used for the determination of two antichagasic drugs in human plasma: nifurtimox and benznidazole. The effects of experimental parameters on extraction efficiency-the type and volume of ionic liquid and disperser solvent, pH, nature and concentration of salt, and the time for centrifugation and extraction-were investigated and optimized. Matrix effects were detected and thus the standard addition method was used for quantification. This microextraction procedure yielded significant improvements over those previously reported in the literature and has several advantages, including high inter-day reproducibility (relative standard deviation=1.02% and 3.66% for nifurtimox and benznidazole, respectively), extremely low detection limits (15.7 ng mL(-1) and 26.5 ng mL(-1) for nifurtimox and benznidazole, respectively), and minimal amounts of sample and extraction solvent required. Recoveries were high (98.0% and 79.8% for nifurtimox and benznidazole, respectively). The proposed methodology offers the advantage of highly satisfactory performance in addition to being inexpensive, simple, and fast in the extraction and preconcentration of these antichagasic drugs from human-plasma samples, with these characteristics being consistent with the practicability requirements in current clinical research or within the context of therapeutic monitoring.

[Massive haemorrhage after bivalirudin anticoagulation in two heart transplant patients]. / Hemorragia masiva después de anticoagulación con bivalirudina en 2 casos de pacientes con trasplante cardiaco.

Heparin-induced thrombopenia is a common autoimmune complication. It is a prothrombotic state due to the formation of antibodies against heparin/platelet factor 4 complexes. In this situation drugs other than heparin must be used for anticoagulation during extracorporeal circulation (bypass) surgery. Two cases of heart transplantation are presented in whom bivalirudin was used as an anticoagulant during the cardiopulmonary bypass. Severe bleeding complications were observed in both patients. The diagnosis of heparin-induced thrombopenia needs to be improved, as well as the development of protocols for using new drugs other than heparin. For this reason, we have reviewed current protocols and alternative therapies to heparin.

Correlation of immunological markers with graft vasculopathy development in heart transplantation.

This study examined the imbalance between T effector cells (Th1 defined as CD3+ interferonγ+) and T regulatory cells (Treg defined as CD4+CD25(high)FoxP3+) as a valuable albeit limited marker of cardiac allograft vasculopathy (CAV) after heart transplantation (HTx). CAV remains, with neoplasms, the most important cause of death in patients surviving the first year after HTx. It is an immune-mediated pathology, although nonimmune factors may also play a role. The process included concentric fibrous intima hyperplasia that narrows the entire length of the affected arteries. Coronary angiography is the usual method of diagnosis. Because a transplanted heart is a denervated organ, CAV is not diagnosed until the disease reaches an advanced stage, in which case transplantation is the only option for treatment. Although the host's immune response against an allogeneic graft is the major cause of endothelial dysfunction, the objective of this study was to detect anti-allogeneic responses on peripheral blood, seeking to identify signs of CAV before classical methods to predict outcomes in HTx recipients. CD3, CD4, CD8, CD19, CD56, Th1, and the Treg mononuclear cell populations were studied in 37 de novo and 20 long-term (more than 3 years) HTx patients as well as 20 healthy volunteers using flow cytometry. A progressive increase in CD8 and Th1 percentages and decrease in the CD4 population were detected during follow-up. Although Th1 changes also reflect processes not related to CAV receiver operating characteristics analysis of Th1/Treg ratio showed an area under the curve of 0.976, with an estimated sensitivity of 100% and specificity of 90%. The positive prediction value was 58.8% and the negative prediction value, 100%. These results prove that the Th1/Treg ratio was an important marker to following host immune response after HTx. The results confirm the need to test other T lymphocyte subsets.

Assessment of immunological markers as mediators of graft vasculopathy development in heart transplantation.

Heart transplantation (HT) remains the treatment of choice for patients with end-stage heart failure. Cardiac allograft vasculopathy (CAV), a diffuse form of coronary atherosclerosis, is the major cause of death after the first year of HT. CAV is thought to be multifactorial in origin. Although nonimmune factors may play a role in CAV development, it is primarily an immune-mediated disease. CAV is diagnosed by routine annual coronary angiography, and usually when diagnosed, the disease is advanced. There is a need to develop noninvasive surrogate markers for early detection. For this purpose, careful immune monitoring and graft histologic assessment are mandatory. The main objective of this study was the assessment of immunologic markers as mediators of CAV development in HT. Flow cytometry was performed to assess peripheral blood mononuclear cell populations forming CD3, CD4, CD8, CD19, CD56, Th1 (CD3+IFNγ+) or Treg (CD4+CD25(high)FoxP3+) markers among 20 de novo HT recipients. The control group included 13 patients who were more than 2 years post-HT (four with and nine without CAV) as well as 20 healthy subjects. CAV-related events over 2 years' follow-up correlated with the Th1/Treg ratio. An increased Th1 lymphocyte percentage was detected over the follow-up. Patients with medium and high Th1/Treg ratios showed higher acute rejection scores as well as greater incidences of CAV. These results indicated that the Th1/Treg ratio may represent a valuable marker to monitor allospecific T-cell responses in peripheral blood. Changes in the Th1/Treg ratio may help in the early detection of patients at risk for CAV. More studies with longer follow-up are needed to confirm these preliminary results.

Inferior vena cava obstruction by a cardiac mass: unusual presentation of primary antiphospholipid syndrome.

We present a previously healthy 43-year-old woman with a cardiac mass that caused almost total occlusion of the inferior vena cava at its junction with the right atrium. The resected mass proved to be a thrombus. Preoperative imaging tests could not distinguish it from other intracardiac tumors such as myxoma. A postoperative immunological study revealed a primary antiphospholipid syndrome. This case presents an unusual diagnosis of antiphospholipid syndrome and a rare location of a cardiac thrombus.

[Severe abdominal complications in patients treated with heart transplantation: the problem of clinical inexpressiveness]. / Complicaciones abdominales graves en los pacientes sometidos a trasplante cardíaco: el problema de la inexpresividad clínica.

INTRODUCTION: In transplanted patients, immunosuppressive drugs can mask habitual pathologies that impede their diagnoses and management. Abdominal pathology gives up to 2-20%, 50% of which is surgical, with a mortality of 10-40%. The most frequently detected pathologies are: acute pancreatitis, peptic ulceration and intestinal obstruction. OBJECTIVES: To determine the alarm parameters, more adequate diagnostic procedures and the most frequent causes of morbidity and mortality in order to attempt to avoid them. METHODS: In our center 225 heart transplantations were performed from May 1984 to October 1997. The severe abdominal complications, time of appearance, implication of immunosuppressive drugs and presence of rejection were studied in these patients. RESULTS: 35 severe abdominal complications were detected (incidence 12.9%), with the majority differing (> 1 year following transplant). The most frequently detected pathologies were digestive hemorrhages and perforations. Acute pancreatitis was 11%. The immunosuppressive drugs used were prednisona, cyclosporin and azathioprine. In 12 out of 29 patients the abdominal complication was in the context of acute rejection. CONCLUSIONS: Even with some non-specific abdominal symptoms in these group of patients it is important to rule out severe pathologies such as acute pancreatitis or empty viscera perforation. The detection of amylases and lypases in the blood and an echographic or tomographic abdominal study should be performed early with a digestive hemorrhage it is important to perform an endoscopy. If the surgical intervention seems imminent it's better to perform it without any delay, because it has been demonstrated that the delay is worse than the probable rejection.

Burden of myocardial damage in cardiac allograft rejection: scintigraphic evidence of myocardial injury and histologic evidence of myocyte necrosis and apoptosis.

BACKGROUND: Because myocardial damage determines morbidity and outcomes in heart transplant rejection, assessment of total burden of myocardial damage is highly desirable. In addition to myocyte necrosis, programmed cell death, or apoptosis, has recently been shown to contribute to cardiac allograft rejection. In the present study, we noninvasively determined myocardial damage by antimyosin scintigraphy and compared it with necrotic and apoptotic myocardial damage in endomyocardial biopsy (EMB) specimens. METHODS AND RESULTS: Forty scintigraphic and histologic studies were simultaneously performed. Of these, 19 patients had no EMB evidence of allograft rejection (group I, International Society of Heart and Lung Transplantation [ISHLT] grade 0/4), 12 had mild rejection (group II, ISHLT grades 1A and 1B), and 9 had evidence of moderate allograft rejection (group III, ISHLT grades 2, 3A, and 3B). None of the biopsies demonstrated severe allograft rejection (ISHLT grade 4/4). The severity of global myocyte damage in 40 patients was assessed by antimyosin scintigraphy. Endomyocardial biopsies were performed in these patients within 48 hours of imaging study; biopsy specimens were characterized for presence of myocyte necrosis and apoptosis. Evidence of myocyte necrosis was observed in 9 (23%) of 40 EMB specimens. Nineteen EMB specimens of group I had no inflammation and no myocyte necrosis, 12 of group II specimens showed interstitial mononuclear cell infiltration (only) but no myocyte necrosis, and all 9 of group III specimens had evidence of cellular infiltration and myocyte damage. Myocyte necrosis was assessed by hematoxylin-eosin and trichrome staining of EMB specimens. On the other hand, apoptosis of myocytes, as assessed by TUNEL staining of DNA fragments, was seen in 22 (55%) of the 40 biopsy specimens: 47%, 58%, and 67% in groups I, II and III, respectively. Abnormal antimyosin scan findings, indicating presence of myocardial damage, were observed in 9 of the 19 patients in group I and in all patients in groups II and III. Although positive antimyosin scan results in group III patients are concordant with the presence of histologic myocardial necrosis, myocardial uptake of antimyosin antibodies in groups I and II (no apparent myocyte damage at light microscopic examination) could reflect either sampling error of the biopsy or ongoing apoptotic myocyte damage. CONCLUSIONS: Apoptosis of myocytes is frequently observed during cardiac allograft rejection. The presence of apoptotic myocytes in the absence of histologic rejection activity in patients with antimyosin uptake suggests that apoptosis could be an additional mechanism of transplant-associated myocardial damage.

Efficacy of augmented immunosuppressive therapy for early vasculopathy in heart transplantation.

OBJECTIVES: The present study was undertaken to prospectively and comparatively evaluate the role of serial myocardial perfusion imaging and coronary angiography for the detection of early vasculopathy in a large patient population and also to determine the short- and long-term efficacy of augmented immunosuppressive therapy in the potential reversal of the early vasculopathy. BACKGROUND: Allograft vasculopathy is the commonest cause of death after the first year of heart transplantation. Anecdotal studies have reported the efficacy of augmented immunosuppressive therapy after early detection of vascular involvement. However, no prospective study has evaluated the feasibility of early detection and treatment of allograft vasculopathy. METHODS: In 76 cardiac allograft recipients, 230 coronary angiographic and 376 scintigraphic studies were performed in a follow-up period of 8 years. Angiography was performed at 1 month and every year after transplantation, and thallium-201 scintigraphy at 1, 3, 6 and 12 months after transplantation and twice a year thereafter. Prospective follow-up of 76 patients showed that 18 developed either angiographic or scintigraphic evidence of coronary vasculopathy. All episodes were treated with 3-day methylprednisolone pulse and antithymocyte globulin. RESULTS: Twenty-two episodes of vasculopathy were diagnosed and treated in these 18 patients. Of these 22 episodes, two were detected only by angiography, seven by both angiography and scintigraphy, four by scintigraphy and histologic evidence of vasculitis and nine episodes only by thallium-201 scintigraphy studies. Angiographic and/or scintigraphic resolution was observed in 15 of the 22 episodes (68%) with augmented immunosuppression. The likelihood of regression was higher when treatment was instituted within the first year of transplantation (92%) than after the first year (40%) (p = 0.033). Eighty percent of patients who responded to follow-up. CONCLUSIONS: The present study suggests that early detection of allograft coronary vasculopathy is feasible with surveillance myocardial perfusion or coronary angiographic studies. When identified early after transplantation, immunosuppressive treatment may result in regression of coronary disease.

[Intramural hematomas of the ascending aorta]. / Hematomas intramurales en aorta ascendente.

We present four patients with intramural hematomas in the ascending aorta. Diagnostic suspicion was aortic dissection in two of them. Prompt surgical procedures were performed in all of them. After reviewing other series, we conclude that ascending aorta hematomas should be treated as true aortic dissections.

Assessment of the appropriateness of the decision of heart transplantation in idiopathic-dilated cardiomyopathy.

One hundred thirty patients with idiopathic-dilated cardiomyopathy were referred for heart transplantation to our center and followed for 18 months. Heart transplantation was performed on 63 patients, 17 patients died before transplantation due to heart failure, and 50 patients never had transplantation. Clinical, electrocardiographic, echocardiographic, and hemodynamic data of the 50 nontransplanted survivors and the 17 patients who died were used to identify independent risk variables with discriminant analysis. Using a statistical model based on the results of discriminant analysis, each of the remaining 63 transplanted patients were predicted as being alive or dead in absence of transplantation. The discriminant analysis identified right atrial pressure, cardiac index, and the New York Heart Association functional class as the strongest predictors of 18-month outcome. The accuracy of the model in predicting survival without transplantation in the nontransplanted group of patients, based on the concordance between actual and predicted outcome, was 85% (kappa = 0.62). Subsequent application of this model to the transplanted group of patients suggested that the decision for transplantation was appropriate in 41 of the 63 patients, and could have been premature in the remaining 22 patients predicted as alive. These results suggest that two-thirds of patients receiving transplants would have died without intervention, but the decision to transplant could have been premature in the remaining patients.

[Minimally invasive aortic valve replacement]. / Sustitución valvular aórtica mínimamente invasiva.

INTRODUCTION: Minimally invasive surgery is being applied to certain procedures in cardiac surgery. Aortic valve replacement presents the highest number of cases in which this approach is feasible. MATERIAL AND METHODS: Fifteen patients, aged 16 to 75 years, underwent aortic valve replacement through a 10 cm incision at the level of the second intercostal space. Cardiopulmonary bypass was instituted through cannulation of the aorta and the femoral vein. RESULTS: Adequate exposure of the aortic root was achieved in all cases. Valve replacement was accomplished with a mean ischemic time of 50 +/- 6 minutes and a pump time of 80 +/- 14 minutes. Mean chest drainage was of 310 +/- 251 ml. The patients were discharged between the third and the fifth day of the postoperative course. CONCLUSIONS: A transverse incision at the level of the second intercostal space provides an excellent exposure for aortic valve replacement. Surgical times are not excessively prolonged and patient's recovery is faster and less painful than with the standard midline sternotomy.

The Spanish Monostrut Study Group: a ten-year experience with 8,599 implants.

BACKGROUND: The Monostrut valve is a pyrolytic carbon, tilting-disc prosthesis with no welds. After the first implantation in Spain in May 1983, the Spanish Monostrut Study Group was established to evaluate prospectively the performance of the valve using uniform protocols. METHODS: During a 10-year period, 8,599 Monostrut valves were implanted in 7,317 patients in 22 centers. Mean age was 53.3 +/- 11 years. Of the total group, 3,229 underwent aortic valve replacement, 2,806 had mitral valve replacement, and 1,282 had double valve replacement. Follow-up was 96% complete, with a mean period of 4.3 years and a total of 29,155 patient-years. RESULTS: The operative mortality rate was 7.2%. The 10-year probability of freedom from valve-related complications and linearized rates (event/100 patient-years in parentheses) were as follows: structural deterioration, 100% (0); nonstructural dysfunction, 96% +/- 0.5% (0.51); thromboembolism, 82.9% +/- 1.5% (1.32); anticoagulant-related hemorrhage, 87.6% +/- 1.4% (0.98); and prosthetic valve endocarditis, 96.1% +/- 0.5% (0.48). There were five obstructive valve thromboses (0.017/100 patient-years). Actuarial freedom from reoperation was 95% +/- 0.5% at 10 years. Actuarial freedom from all valve-related morbidity was 70.2% +/- 1.6%; freedom from all valve-related morbidity and mortality (including operative and sudden deaths) was 62.6% +/- 2% (70.1% +/- 2% for aortic valve replacement, 56.9% +/- 2% for mitral valve replacement, and 59.8% +/- 3% for double valve replacement; p < 0.0001). Clinically, 5,988 patients (94%) are in New York Heart Association class I-II. CONCLUSIONS: The Monostrut valve has shown no structural failures and a low rate of valve-related complications over a 10-year period in a large patient population.

[Peritoneal migration of defibrillator implanted behind the rectus abdominis]. / Migración peritoneal de desfibrilador implantado tras recto abdominal.

Automatic Implantable Cardioverter Defibrillator (AICD) has become a therapeutic option to malignant ventricular tachyarrhythmias. Its bulky device responds for discomfort and the likelihood of generator extrusion or migration, when implanted under the subcutaneous tissue. Among alternative sites, pre-peritoneal location has several advantages but hides its own risks. We present a generator peritoneal migration from a pocket made behind the rectus abdominis.

[Etiologic and pathologic study of respiratory disease in lambs from intensive breeding facilities in southern Spain]. / Estudio etiologico y lesional de las afecciones respiratorias en corderos criados de forma intensiva en sur de España.

Between 1991 and 1993, it was observed epidemiologically that respiratory disturbances in lambs are associated with high temperatures during the summer. The etiological agent isolated is principally Mycoplasma ovipneumoniae; moreover, Actinobacillus pleuropneumoniae biovar A has been isolated in a high number of samples. Histopathologically, an interstitial bronchopneumonia was the main lesional finding; this lesion is associated with previous mycoplasma infection.

Doppler-derived gradients in normally functioning Monostrut Björk-Shiley prostheses.

In summary, reference values of Doppler gradients obtained in a large number of patients with normal-functioning mitral and aortic Monostrut Björk-Shiley prostheses are reported. It is shown that the value of the transprosthetic gradient increases with decreasing valve size in patients with aortic prostheses. No individual significant variations of the transprothetic Doppler gradient during a 3-year follow-up were observed.

[The diagnosis of rejection activity in the heart transplant by monoclonal antimyosin antibodies]. / Diagnóstico de la actividad de rechazo en el trasplante cardíaco mediante anticuerpos monoclonales antimiosina.

A novel noninvasive sensitive mean to detect cardiac rejection is described: myocardial uptake of 111In-labeled monoclonal antimyosin antibodies (MAA). All patients showing rejection at cardiac biopsy disclosed positive MAA studies. However, a large percentage of positive studies in the presence of negative biopsies were detected. This discrepancy can be ascribed to a false-negative biopsy result. During the first year posttrasplantation MAA studies are useful to predict severe rejection-related complications, but due to high sensitivity of MAA, treatment for rejection in this period must be based on biopsies, as criterium to treat for rejection on the basis of MAA scans would lead to excessive immunosuppression. After the first year of transplantation, individual patient management can ben implemented on the basis of risk stratification using MAA scans: Negative MAA scans entail an almost nil probability of detecting rejection during long-term follow-up (low-risk group), whereas positive MAA scans imply a probability of detecting near 1 episode of rejection and requirement for treatment per year. In summary, at our institution biopsies are avoided as from the first year after transplantation; after such period, MAA scans allow risk stratification and treatment for rejection based on the results of MAA scans in individual patients.

[A report of a rare case of primary angiosarcoma of left atrium and a review of the literature]. / Presentación de un caso raro de angiosarcoma primario de aurícula izquierda y revisión de la literatura.

A 66[correction of 60]-year-old female patient was admitted to hospital for clinical signs of pericardial tamponade. The pericardiocentesis revealed an hematic effusion and a left appendage mass was diagnosed by transesophageal echocardiography. At surgery, a left atrial tumour was resected which histological examination showed to be an undifferentiated angiosarcoma-endothelioma with difficult histological classification. The tumoral screening was negative and the patient was discharged from the hospital. Seven months later the patient was readmitted for two parasternal tumours which anatomopathologic study revealed to be subcutaneous metastases. The rare localization in the left atrium and subcutaneous metastatic spread were discussed.

[Cardiovascular surgery in Spain in 1992. The Registry of Operations of the Spanish Society of Cardiovascular Surgery (SECCV)]. / Cirugía cardiovascular en España en el año 1992. Registro de operaciones de la Sociedad Española de Cirugía Cardiovascular (SECCV).

The Spanish Society of Cardiovascular Surgery Registry of 1992 includes data from 41 centers. Within this year a total of 24,127 patients were operated on, with an average of 588 operations/center. Twelve thousand twenty-two of these were cardiac operations under extracorporeal circulation, with an average of 300 cases/hospital. The average of cardiac surgeons/hospital was 5.9 and the average of open heart operations/surgeon was 50. For the first time, the number of coronary bypass surgeries was superior to that of valvular procedures (5,049 vs 4,951). In the coronary bypass patients the average of grafts/patient was 2.43. The number of valvular prostheses implanted was 5,526 and 81% of these were mechanical. The number of patients operated on for congenital cardiac defects was 2,251 (1,278 open heart surgeries and 973 closed). The global mortality in the patients operated on under extracorporeal circulation was 7.6% (7.0% in valvular, 7.9% in congenital and 5.4% in coronary bypass). There were 6,054 patients subjected to surgery for peripheral vascular disease.