Silvana Giuliatti

nopsin, the alkaloidal component of the fire ant (Solenopsis invicta), is a naturally oc‐ curring inhibitor of phosphatidylinositol-3-kinase signaling and angiogenesis. *Blood*,

[246] Sonoda, Y., Hada, N., Kaneda, T., Suzuki, T., Ohshio, T., Takeda, T., & Kasahara, T. (2008). A synthetic glycosphingolipid-induced antiproliferative effect in melanoma cells is associated with suppression of FAK, Akt, and Erk activation. *Biological and*

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

109(2), 560-565.

Applications

72

*Pharmaceutical Bulletin*, 31(6), 1279-1283.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/ 52334


**4. Conclusion**

There are evolving global challenges on the utilization of non-renewable resources in the manufacturing industry and increasingly stringent environmental legislation. Both consumers and regulatory agencies are thriving for products that reduce dependency on fossil fuels and thus, are more environmentally friendly. As such, this paves for an opportunity to embrace the use of natural fibers in products and composites leading to significant growth of biobased economy, which the present chapter intends to stimulate.

The field of study of plant fibers that can be industrially exploited remains open. In this chapter, a particular emphasis has been put on their production, in particular on the methods that are generally used to separate them from their originating plants. To date, the question of improving the quality of the extracted fiber has been satisfactorily answered, particularly as regards the possibility of combining several methods when necessary. Some other questions still require research. These include, among others, growing conditions for seed multiplication and fiber production, harvesting methods, optimisation of fiber separation, the molecular basis for improving fiber decortication and performance. The knowledge gained from this work could be used to design new varieties of fibers, tailored for specific industrial applications. Similarly, the recourse to proteomics [68, 69], to isolate genes involved in the biosynthesis of cell wall lignin and hemicellulose in tobacco. Variations in these constituents can affect the fiber quality and cellulose availability. This could then lead to a new orientation on molecular selection research as well as genetic modifications studies to improve the quality of plant fibers.

Morphology and surface behavior of plant fibers are studied using various techniques such as XRD, FTIR, SEM, AFM, TEM and thermogravimetric analysis that helps in understanding the nature of natural fibers.

In terms of the mechanical behavior of plant fibers, important milestones have been achieved to highlight the influence of the chemical composition and structural parameters of the plant wall on their tensile properties. The microstructure of plant fibers is very complex, precisely when it comes to defining generalizable geometric and analytical models that describe it. As mentioned above, improving the mechanical properties of fibers may require the introduction of new types of fibers. And we could mention in this regard the ongoing research on spinning with solvents [70, 71], to obtain fibers of greater strength and low scattered properties. Understanding how fiber morphology affects the properties of composite materials is essential. More precisely, it is important for the selection of new fibers and for the cultivation of fibrous plants genetically selected. This would help to predict their potential for reinforcement in other materials to achieve desired properties.

Investigation of the viscoelastic properties of plant fibers has also been outlined. A variety of dynamic modulus measurement methods exists including ultrasonic wave propagation and the flexural resonance method presented here, for which normal modes of vibration are monitored. Stress relaxation tests are to be carried out to retrieve stress over time as well as the elastic modulus of the fiber material. A mathematical method for extracting the relaxation modulus from relaxation experimental data has to be proposed to this end. Proper selection of the testing vibrational mode and machine cross-head speed (during relaxation) appear important in the suggested methods in order to avoid dispersive results. The Young's modulus that is obtained from the dynamic behavior of the specimen should, therefore, reflects the frequency dependence of the material.


*Article* **Sustained Inflation Reduces Pulmonary Blood Flow during Resuscitation with an Intact Cord**

**Jayasree Nair 1,\* , Lauren Davidson 1,2, Sylvia Gugino 1 , Carmon Koenigsknecht 1 , Justin Helman 1 , Lori Nielsen 1 , Deepika Sankaran 3 , Vikash Agrawal 1,4, Praveen Chandrasekharan 1 , Munmun Rawat 1 , Sara K. Berkelhamer 1,5 and Satyan Lakshminrusimha 3**

**Abstract:** The optimal timing of cord clamping in asphyxia is not known. Our aims were to determine the effect of ventilation (sustained inflation–SI vs. positive pressure ventilation–V) with early (ECC) or delayed cord clamping (DCC) in asphyxiated near-term lambs. We hypothesized that SI with DCC improves gas exchange and hemodynamics in near-term lambs with asphyxial bradycardia. A total of 28 lambs were asphyxiated to a mean blood pressure of 22 mmHg. Lambs were randomized based on the timing of cord clamping (ECC—immediate, DCC—60 s) and mode of initial ventilation into five groups: ECC + V, ECC + SI, DCC, DCC + V and DCC + SI. The magnitude of placental transfusion was assessed using biotinylated RBC. Though an asphyxial bradycardia model, 2–3 lambs in each group were arrested. There was no difference in primary outcomes, the time to reach baseline carotid blood flow (CBF), HR ≥ 100 bpm or MBP ≥ 40 mmHg. SI reduced pulmonary (PBF) and umbilical venous (UV) blood flow without affecting CBF or umbilical arterial blood flow. A significant reduction in PBF with SI persisted for a few minutes after birth. In our model of perinatal asphyxia, an initial SI breath increased airway pressure, and reduced PBF and UV return with an intact cord. Further clinical studies evaluating the timing of cord clamping and ventilation strategy in asphyxiated infants are warranted.

**Keywords:** sustained inflation; delayed cord clamping; placental transfusion; pulmonary blood flow; perinatal asphyxia

### **1. Introduction**

Birth asphyxia affects 4 million newborn infants worldwide each year [1,2]. Appropriate resuscitative measures result in a substantial reduction in birth asphyxia-associated mortality and morbidity [3]. Establishing early and effective ventilation is key to successful neonatal resuscitation. Every 30 s delay in the initiation of ventilation increases the risk for early death and morbidity [4]. Sustained inflation (SI) may facilitate the early establishment of functional residual capacity (FRC). Revised European resuscitation guidelines recommend maintaining inflation pressure for 2–3 s for the first five inflations in apneic infants [5]. Minimal evidence exists for its use in term infants. A single SI immediately after birth improved the speed of circulatory recovery and lung compliance in the resuscitation of near-term asphyxiated lambs [6]. SI with continuous chest compressions is being evaluated as a resuscitation strategy in animal studies [7,8].

**Citation:** Nair, J.; Davidson, L.; Gugino, S.; Koenigsknecht, C.; Helman, J.; Nielsen, L.; Sankaran, D.; Agrawal, V.; Chandrasekharan, P.; Rawat, M.; et al. Sustained Inflation Reduces Pulmonary Blood Flow during Resuscitation with an Intact Cord. *Children* **2021**, *8*, 353. https:// doi.org/10.3390/children8050353

Academic Editor: Simone Pratesi

Received: 7 April 2021 Accepted: 27 April 2021 Published: 29 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Available literature on SI is in neonatal/animal models with immediate or early cord clamping (ECC). ECC in asphyxia compromises both sources of LV preload (pulmonary venous (PV) return and umbilical venous (UV) return through the foramen ovale), resulting in decreased perfusion [9]. However, because of a lack of data demonstrating benefits and limitations of performing extensive resuscitation with an intact cord, delayed cord clamping (DCC) is currently not recommended, but is being evaluated in randomized controlled trials (e.g., SAVE NCT04070560). There is little information on resuscitation with SI and an intact cord. Our objectives were to determine the effect of ventilation (SI vs. positive pressure ventilation—PPV) with early or delayed cord clamping on resuscitation parameters in asphyxiated, bradycardic, near-term lambs. We hypothesized that SI with DCC would result in improved pulmonary and cerebral hemodynamics and enhanced gas exchange.

#### **2. Materials and Methods**

Surgical Protocol: The study was approved by the Institutional Animal Care and use committee (IACUC) at the University at Buffalo and the methods were consistent with the NIH guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978) and in accordance with the ARRIVE guidelines. Time-dated ewes at 142–145d gestation (Term 147d) from May Family Enterprises (Buffalo Mills, PA, USA) were fasted overnight, then induced for anesthesia with intravenous diazepam and ketamine. Ewes were intubated and ventilated with 2–3% isoflurane. In total, 28 fetal lambs were partially exteriorized, intubated and drained of excess lung fluid to simulate the decrease in lung liquid with labor. ETT was occluded to prevent air exchange with gasping. Fetal lambs were instrumented [10] with catheters placed in the right carotid artery (CA) and jugular vein (JV) for blood pressure measurements and blood sampling. Flow probes (Transonic Systems Inc., Ithaca, NY, USA) were placed around the left CA, left pulmonary artery and one umbilical artery and vein. Preductal pulse oximetry was measured using a Nonin pulse oximeter sensor (EQUANOX™, Nonin Medical Inc, Plymouth, MN, USA). Asphyxia was induced by umbilical cord occlusion using a vascular occluder until the mean blood pressure (MBP) was less than or equal to 22 mmHg, when the cord was released, and lamb was exteriorized. In a prior study, the target MBP < 20 mmHg resulted in a need for extensive resuscitation and high mortality [6]. By initiating resuscitation at a MBP of 22 mmHg, we anticipated an adequate degree of asphyxia without extensive mortality or the need for advanced resuscitative efforts.

Randomization: Lambs were randomized prior to delivery to ECC (cord clamped immediately) and DCC (cord was clamped after 60 s). They were further randomized based on the timing of ventilation onset as well as the type of initial ventilation into five groups: ECC + V, ECC + SI, DCC, DCC + V and DCC + SI (Figure 1).

Resuscitation: A T-piece resuscitator was used for ventilation, with 21% O2 at a rate of 40 breaths/min, an initial peak inflation pressure (PIP) of 35 cm H2O and a peak endexpiratory pressure (PEEP) of 5 cm H2O during the intervention period for all groups. The two SI groups—ECC + SI and DCC + SI—were resuscitated with an initial SI breath with a T-piece resuscitator with a PIP of 35 cm H2O for 30 s. In the DCC group, ventilation was delayed until after the cord was clamped at 60 s. The intervention period was defined as the 30 s period when SI breath was delivered. In non-SI groups, the corresponding 30 s time period after the onset of resuscitation was taken as the intervention period. PIP was subsequently adjusted as needed to obtain an adequate chest rise, and PPV continued with 21% O2 according to neonatal resuscitation guidelines [11] at a rate of 40–60 breaths/min in all groups. If lambs went into asystole, resuscitation was continued with CC and 100% oxygen [11]. Epinephrine (0.01–0.03 mg/kg/dose) was administered intravenously at 3 min for asystolic lambs. Inspired oxygen was titrated to maintain the recommended preductal SpO2 [11]. Arterial blood gases were drawn at the initiation of resuscitation, then every minute for the first 5 min followed by every 5 min until the end of the study, and analyzed using a radiometer blood gas analyzer (ABL 800 FLEX, Copenhagen, Denmark).

Prespecified hemodynamic parameters were recorded for a period up to 30 min from birth, following which the lambs were euthanized using pentobarbital per approved lab protocols. Physiological parameters including heart rate (HR), systemic BP and vascular flows were continuously recorded using AcqKnowledge Acquisition and Analysis Software (BIOPAC systems, Goleta, CA, USA). An end-tidal carbon dioxide adapter was attached to the ETT and a Philips NM3 monitor (Respironics, Murrysville, PA, USA) was used to measure airway pressure. *Children* **2021**, *8*, x FOR PEER REVIEW 3 of 12

**Figure 1.** Study groups based on the timing of cord clamping and type of ventilation. DCC: Delayed cord clamping, ECC: Early cord clamping, SI: Sustained inflation, PPV: Positive pressure **Figure 1.** Study groups based on the timing of cord clamping and type of ventilation. DCC: Delayed cord clamping, ECC: Early cord clamping, SI: Sustained inflation, PPV: Positive pressure ventilation.

ventilation Resuscitation: A T-piece resuscitator was used for ventilation, with 21% O2 at a rate of 40 breaths/min, an initial peak inflation pressure (PIP) of 35 cm H2O and a peak endexpiratory pressure (PEEP) of 5 cm H2O during the intervention period for all groups. The two SI groups—ECC + SI and DCC + SI—were resuscitated with an initial SI breath with a T-piece resuscitator with a PIP of 35 cm H2O for 30 s. In the DCC group, ventilation was delayed until after the cord was clamped at 60 s. The intervention period was defined as the 30 s period when SI breath was delivered. In non-SI groups, the corresponding 30 s time period after the onset of resuscitation was taken as the intervention period. PIP was subsequently adjusted as needed to obtain an adequate chest rise, and PPV continued with 21% O2 according to neonatal resuscitation guidelines [11] at a rate of 40–60 breaths/min in all groups. If lambs went into asystole, resuscitation was continued with CC and 100% oxygen [11]. Epinephrine (0.01–0.03 mg/kg/dose) was administered intravenously at 3 min for asystolic lambs. Inspired oxygen was titrated to maintain the The magnitude of placental transfusion was assessed using biotinylated RBC to measure the red cell volume [12]. Next, 20 mL of maternal packed RBC was drawn a few days prior to the study and washed with a buffer solution three times. This was then divided into two groups; a low-density (LD) group of 12 ug/mL biotin and high-density (HD) group of 96 ug/mL biotin, both prepared with a target Hct of 50%. A baseline complete blood count (CBC) was drawn from the fetal lamb before injection. The lowdensity sample was infused into the lamb while still under maternal-fetal circulation, and samples were collected before and 10 min after injection to permit equilibration. This sample was used to measure the fetoplacental RBC volume. After delivery, the high-density biotin-labeled RBCs were injected to measure the newborn RBC volume. FICT-avidin (fluorescent marker) was added to the saved in vivo samples and measured with a flow cytometer. This labeled and counted the number of LD- and HD-labeled RBCs circulating in each sample to help determine the fetal vs. newborn total blood volume for each group studied.

recommended preductal SpO2 [11]. Arterial blood gases were drawn at the initiation of resuscitation, then every minute for the first 5 min followed by every 5 min until the end The magnitude of placental transfusion was assessed between groups using the following equations.

carbon dioxide adapter was attached to the ETT and a Philips NM3 monitor (Respironics, Murrysville, PA, USA) was used to measure airway pressure. The magnitude of placental transfusion was assessed using biotinylated RBC to Primary end-points: The primary end-point was the time to circulatory stabilization, defined as the time taken to reach the baseline carotid blood flow—CBF (prior to umbilical cord occlusion), stable HR (≥100/min) and a MBP of ≥40 mmHg.

measure the red cell volume [12]. Next, 20 mL of maternal packed RBC was drawn a few days prior to the study and washed with a buffer solution three times. This was then divided into two groups; a low-density (LD) group of 12 ug/mL biotin and high-density Secondary end-points: The secondary end-points included changes in systemic hemodynamics (CBF and MBP), left pulmonary blood flow (PBF) and umbilical arterial (UA) and venous (UV) flow changes during the 30 s intervention period between groups.

(HD) group of 96 ug/mL biotin, both prepared with a target Hct of 50%. A baseline complete blood count (CBC) was drawn from the fetal lamb before injection. The lowdensity sample was infused into the lamb while still under maternal-fetal circulation, and

sample was used to measure the fetoplacental RBC volume. After delivery, the highdensity biotin-labeled RBCs were injected to measure the newborn RBC volume. FICTavidin (fluorescent marker) was added to the saved in vivo samples and measured with a flow cytometer. This labeled and counted the number of LD- and HD-labeled RBCs

Statistical analysis and power calculation: Power and sample size calculations were based on a previous study of SI in asphyxiated near-term lambs [6], where the time to achieve circulatory stabilization within subject group was normally distributed with a standard deviation of 12 s. If the true difference in SI and PPV means was 25 s, we needed to study five subjects in each group to be able to reject the null hypothesis with a probability (power) of 0.8 and a Type I error probability of 0.05. Continuous variables were expressed as the mean and standard deviation (SD) and analyzed by ANOVA between groups with Fisher's post hoc test. Categorical variables were analyzed using the chi-square test with Fisher's exact test if appropriate. GraphPad Prism (San Diego, CA, USA) was used for statistical analysis. Statistical significance was defined as *p* < 0.05.

#### **3. Results**

Lambs were comparable in birth weight with similar baseline hemodynamic measurements that were obtained after instrumentation. The baseline UV flow was higher than the UA flow in most lambs. The extent of asphyxia was similar, as reflected by comparable pH and lactate levels (Table 1). Although intended as a model of bradycardia with perinatal asphyxia without cardiac arrest, 2–3 lambs in each group progressed to asystole similar to previously described studies in this model with cord occlusion [13].

#### *3.1. Airway Pressure*

As expected, the mean airway pressures in the SI groups, ECC + SI and DCC + SI, were significantly higher than in the PPV groups, ECC + V and DCC + V (Table 2), during the intervention period.

#### *3.2. Primary Outcomes*

No significant differences were noted among the five groups in time to reach baseline CBF, HR ≥ 100 bpm or MBP ≥ 40 mm Hg (Table 2).

#### *3.3. Hemodynamics during Intervention Period*

Among the DCC groups, the DCC + SI group had significantly decreased PBF compared to the DCC and DCC + V groups. MBP was comparable between groups (Table 2). Umbilical flows during the intervention period in all DCC groups were significantly lower compared to the baseline values prior to the onset of asphyxiation by cord compression. Significantly lower UV flow was seen during SI compared to DCC and DCC + V without any corresponding statistically significant differences in UA flow (Table 2).

**Table 2.** Primary outcomes and hemodynamic parameters during the intervention period.

Values are presented as mean ± SD; \* *p* < 0.05 by Kruskal–Wallis one-way analysis of variance between DCC groups; \*\* *p* < 0.005 by Kruskal–Wallis one-way analysis of variance between groups.

#### *3.4. Post Resuscitation Hemodynamics*

CBF decreased during asphyxia and increased after birth in all groups (Figure 2a), reaching baseline values by 5 min. No statistically significant differences in CBF patterns were noted among the five individual groups. However, ECC + V and ECC + SI appeared to result in a rebound of cerebral blood flow above baseline, while DCC (with and without ventilation) resulted in a steady increase back to baseline values without overshoot. PBF (Figure 2b) in all lambs increased after birth; however, at 1–3 min, the PPV groups (ECC + V and DCC + V) demonstrated a trend towards higher PBF, compared to the SI groups (ECC + SI and DCC + SI). MBP and SBP (Figure 2c,d) were similar in the 30 min period after birth, with figures depicting the first 10 min. *Children* **2021**, *8*, x FOR PEER REVIEW 6 of 12

**Figure 2.** Post resuscitation hemodynamics in the first 10 min after birth in the five study groups: (**a**) carotid blood flow, (**b**) pulmonary blood flow, (**c**) mean blood pressure and (**d**) systolic blood pressure in all five study groups, with dashed lines reflecting the SI groups. Data are represented as mean ± SD. Black circle: ECC + V (*n* = 5), pink square dashed: ECC + SI (*n* = 6), green triangle: DCC (*n* = 5), purple triangle: DCC + V (*n* = 5), purple diamond dashed: DCC + SI (*n* = 7). **Figure 2.** Post resuscitation hemodynamics in the first 10 min after birth in the five study groups: (**a**) carotid blood flow, (**b**) pulmonary blood flow, (**c**) mean blood pressure and (**d**) systolic blood pressure in all five study groups, with dashed lines reflecting the SI groups. Data are represented as mean ± SD. Black circle: ECC + V (*n* = 5), pink square dashed: ECC + SI (*n* = 6), green triangle: DCC (*n* = 5), purple triangle: DCC + V (*n* = 5), purple diamond dashed: DCC + SI (*n* = 7).

significantly higher in the DCC + SI group along with a lower PaCO2. Among the DCC groups, the required FiO2 trended lower, and the P/F ratio was significantly higher in the

Blood gas parameters at 2 min pH 6.9 ± 0.1 6.9 ± 0.1 6.9 ± 0.1 7.0 ± 0.2 6.9 ± 0.1 pCO2 (mmHg) 107 ± 16 110 ± 14 130 ± 24 87 ± 24 92 ± 26 pO2 (mmHg) 29 ± 24 27 ± 6 22 ± 14 29 ± 11 20 ± 8 FiO2 0.5 ± 0.4 0.8 ± 0.3 0.5 ± 0.3 0.5 ± 0.4 0.8 ± 0.4 P/F ratio 106 ± 133 41 ± 24 51 ± 38 79 ± 65 39 ± 38 Lactate (mmol/L) 8.3 ± 1.8 8.7 ± 1.5 10.2 ± 2.4 8.9 ± 1.5 11.7 ± 2.5 Blood gas parameters at 5 min pH 6.9 ± 0.2 6.9 ± 0.1 6.9 ± 0.1 6.9 ± 0.2 7 ± 0.2 pCO2 (mmHg) 102 ± 27 95 ± 11 99 ± 39 90 ± 42 66 ± 36 pO2 (mmHg) 61 ± 51 85 ± 50 37 ± 31 46 ± 30 45 ± 17 FiO2 0.6 ± 0.4 0.7 ± 0.3 0.6 ± 0.3 0.4 ± 0.2 0.5 ± 0.4 P/F ratio 126 ± 132 138 ± 60 93 ± 106 149 ± 110 127 ± 113 Lactate (mmol/L) 8.9 ± 2.3 8.8 ± 1.9 9.4 ± 3.4 9.8 ± 3.8 11.2 ± 3 Blood gas parameters at 10 min pH 7.0 ± 0.2 6.9 ± 0.1 6.9 ± 0.1 6.9 ± 0.2 7.0 ± 0.1 pCO2 (mmHg) 81 ± 15 82 ± 27 83 ± 39 73 ± 34 44 ± 15 \* pO2 (mmHg) 82 ± 36 106 ± 68 52 ± 30 76 ± 49 134 ± 80

**(***n* **= 6) DCC (***n* **= 5) DCC + V**

**(***n* **= 5) DCC + SI (***n* **= 6)**

**ECC + V (***n* **= 5) ECC + SI**

*3.5. Gas Exchange and Lactate*

DCC + SI group (Table 3).

**Table 3.** Gas exchange parameters.

#### *3.5. Gas Exchange and Lactate*

Arterial pH, pCO2, pO2 and lactate were similar between groups at 2, 5 and 10 min, as were PaO2/FiO2 (P/F) ratios at 2 and 5 min. However, at 10 min, P/F ratios were significantly higher in the DCC + SI group along with a lower PaCO2. Among the DCC groups, the required FiO2 trended lower, and the P/F ratio was significantly higher in the DCC + SI group (Table 3).

**Table 3.** Gas exchange parameters.

\* *p* < 0.05 by Kruskal–Wallis test of variance in all groups; # *p* < 0.02 Kruskal–Wallis test of variance in DCC groups.

#### *3.6. Magnitude of Placental Transfusion*

Residual placental volume and newborn blood volume were similar between the ECC and DCC groups (Table 4). Speculating that placental transfusion was interrupted during arrest, we performed a subgroup analysis of non-arrested lambs. The DCC lambs had a significantly higher fraction of fetoplacental volume and a decreased residual placental volume compared to the ECC lambs (Table 4).

**Table 4.** Measurement of blood volume and placental transfusion.

Values are presented as mean ± SD \* *p* < 0.05 by Mann–Whitney U test vs. ECC.

#### *3.7. Mode of Ventilation*

Comparing SI (ECC + SI and DCC + SI) vs. PPV (ECC + V and DCC + V), the CBF patterns were not different (Figure 3a); however, the SI significantly reduced PBF in the first 4 min after birth (Figure 3b). MBP and SBP were similar with PPV or SI (Figure 3c,d). *Children* **2021**, *8*, x FOR PEER REVIEW 8 of 12

**Figure 3.** Post resuscitation hemodynamics in the first 10 min after birth in ventilation groups—effect of sustained inflation (gray squares, N = 13) is compared to positive presScheme 10: (**a**) carotid blood (CBF), (**b**) pulmonary blood flow (PBF), (**c**) mean blood pressure (MBP) and (**d**) systolic blood pressure (SBP). Data are represented as mean ± SD. \* *p* < 0.01 by repeated measures ANOVA. **Figure 3.** Post resuscitation hemodynamics in the first 10 min after birth in ventilation groups—effect of sustained inflation (gray squares, N = 13) is compared to positive pressure ventilation (Black circles, N = 10): (**a**) carotid blood (CBF),(**b**) pulmonary blood flow (PBF), (**c**) mean blood pressure (MBP) and (**d**) systolic blood pressure (SBP). Data are represented as mean ± SD. \* *p* < 0.01 by repeated measures ANOVA.

#### *3.8. Timing of Cord Clamping 3.8. Timing of Cord Clamping*

DCC reduced the CBF overshoot that was noted with ECC during the post resuscitation period (Figure 4a). Both groups had similar PBF, MBP and SBP post resuscitation (Figure 4b–d). DCC reduced the CBF overshoot that was noted with ECC during the post resuscitation period (Figure 4a). Both groups had similar PBF, MBP and SBP post resuscitation (Figure 4b–d).

induced by cord occlusion.

**Figure 4.** Post resuscitation hemodynamics in the first 10 min after birth based on the timing of cord clamping: (**a**) carotid blood flow, (**b**) pulmonary blood, (**c**) mean blood pressure and (**d**) systolic blood pressure. Data are represented as mean ± SD. \* *p* < 0.05 repeated measures ANOVA. **Figure 4.** Post resuscitation hemodynamics in the first 10 min after birth based on the timing of cord clamping: (**a**) carotid blood flow, (**b**) pulmonary blood, (**c**) mean blood pressure and (**d**) systolic blood pressure. Data are represented as mean ± SD. \* *p* < 0.05 repeated measures ANOVA. Black circles: ECC. Grey squares: DCC. **Figure 4.** Post resuscitation hemodynamics in the first 10 min after birth based on the timing of cord clamping: (**a**) carotid blood flow, (**b**) pulmonary blood, (**c**) mean blood pressure and (**d**) systolic blood pressure. Data are represented as mean ± SD. \* *p* < 0.05 repeated measures ANOVA. Black circles: ECC. Grey squares: DCC.

#### **4. Discussion 4. Discussion 4. Discussion**

Black circles: ECC. Grey squares: DCC.

In animal models and infants without asphyxia or with mild/moderate compromise, the benefits of physiological cord clamping (DCC after ventilation onset) are well established [9,14]. In our study, we compared DCC using two modes of ventilation, SI and PPV, which we compared to DCC without ventilation and ECC with immediate ventilation (current standard) in severely asphyxiated lambs. Few studies have evaluated DCC in a non-ventilated asphyxiated animal model. Delayed cord clamping for 60 s with ventilation mitigated post resuscitation cerebral hyperemia, but did not offer other substantial hemodynamic advantages. SI transiently decreased pulmonary perfusion, but led to a better gas exchange by 10 min when used in conjunction with DCC (Figure 5). In animal models and infants without asphyxia or with mild/moderate compromise, the benefits of physiological cord clamping (DCC after ventilation onset) are well established [9,14]. In our study, we compared DCC using two modes of ventilation, SI and PPV, which we compared to DCC without ventilation and ECC with immediate ventilation (current standard) in severely asphyxiated lambs. Few studies have evaluated DCC in a non-ventilated asphyxiated animal model. Delayed cord clamping for 60 s with ventilation mitigated post resuscitation cerebral hyperemia, but did not offer other substantial hemodynamic advantages. SI transiently decreased pulmonary perfusion, but led to a better gas exchange by 10 min when used in conjunction with DCC (Figure 5). In animal models and infants without asphyxia or with mild/moderate compromise, the benefits of physiological cord clamping (DCC after ventilation onset) are well established [9,14]. In our study, we compared DCC using two modes of ventilation, SI and PPV, which we compared to DCC without ventilation and ECC with immediate ventilation (current standard) in severely asphyxiated lambs. Few studies have evaluated DCC in a non-ventilated asphyxiated animal model. Delayed cord clamping for 60 s with ventilation mitigated post resuscitation cerebral hyperemia, but did not offer other substantial hemodynamic advantages. SI transiently decreased pulmonary perfusion, but led to a better gas exchange by 10 min when used in conjunction with DCC (Figure 5).

**Figure 5.** Summary: effects of (**A**) ECC + V—current practice, (**B**) DCC + V and (**C**) DCC + SI in our model of ovine asphyxia **Figure 5.** Summary: effects of (**A**) ECC + V—current practice, (**B**) DCC + V and (**C**) DCC + SI in our model of ovine asphyxia induced by cord occlusion. **Figure 5.** Summary: effects of (**A**) ECC + V—current practice, (**B**) DCC + V and (**C**) DCC + SI in our model of ovine asphyxia induced by cord occlusion.

SI as an initial ventilation strategy for lung recruitment has shown some benefits. A two-fold increase in inflation volume and FRC was described with SI in nine term asphyxiated newborns [15]. SI reduced the need for mechanical ventilation at 72 h [16] in preterm infants. Recently, the well-designed multicenter, randomized, SAIL trial evaluating SI in extremely preterm infants at birth was terminated early with infants in the SI group demonstrating increased early mortality [17], without any clear etiology. Interestingly, 15–20% of infants in both arms underwent 30 s DCC prior to SI, though their outcomes were not reported separately. A unique feature of our study is the evaluation of SI in combination with intact cord resuscitation. Contrary to our hypothesis, we could not demonstrate improved systemic or pulmonary hemodynamic outcomes with SI and DCC in an asphyxiated model. In fact, we report deleterious effects of SI on reducing PBF and UV flow, possibly due to an effect on intrathoracic pressure and/or diaphragmatic excursion [18] with an intact cord during the first few minutes after birth. However, these deleterious effects that we observed with a high pressure of 34.6 ± 3 cm H2O with SI may not be seen with lower pressures in a non-asphyxiated model, with or without an intact cord.

Several different time periods and inflation pressures were evaluated in SI. We chose a 30 s SI breath with a peak inflating pressure (PIP) of ~35 cm H2O and PEEP of 5 cm H2O on the basis of prior studies in a similar model. Klingenberg et al. compared five 3 s inflations or a single 30 s inflation in resuscitation of near-term asphyxiated lambs with subsequent ventilation using inflations at 0.5 s at 60/min for all groups [6]. They noted earlier cardiovascular recovery (time taken to achieve a HR > 120/min) by almost 60 s in the single 30 s sustained inflation group compared to the no SI groups. We could not demonstrate a similar benefit to SI with either early or delayed cord clamping in our study; however, the incidence of cardiac arrest in our model complicated the analysis of this data.

While no differences in gas exchange were noted at 2 and 5 min, we demonstrated differences at 10 min (Table 3). We suspect that SI resulted in better recruitment of the lung establishing FRC, resulting in higher PaO2 values. In addition, a combination of SI and DCC enabled "dual-ventilation" during the first 5 min, leading to normocapnia. Hypercapnia is known to enhance V/Q mismatch [19]. We speculate that lambs in the DCC + SI arm achieved a higher P/F ratio due to better alveolar recruitment and V/Q matching secondary to normocapnia (Table 3).

The interplay between different modes of ventilation and timing of cord clamping has not been evaluated before and is a strength of our study. There is insufficient evidence for or against DCC in infants requiring resuscitation [11]. Currently, most information on intact cord resuscitation in asphyxia comes from translational ovine models similar to our current study. DCC for 15 min restored cardiac output and oxygenation and mitigated postasphyxial rebound hypertension after resuscitation in a non-arrest ovine model of perinatal asphyxia induced by maternal iliac artery ligation [13]. In lambs with asphyxial arrest, DCC for 10 min caused significant reductions in post-asphyxial rebound hypertension, cerebral blood flow and oxygenation [20]. These studies indicate improved hemodynamics with a longer DCC duration, and along with a difference in methodology of induced asphyxia (cord occlusion vs. maternal iliac artery ligation), may explain lack of sustained benefits in our study. With a clinically feasible and widely adapted shorter duration of a DCC of 60 s, we demonstrated the mitigation of post-asphyxial hyperemia in our model.

DCC is a reasonable option for term and preterm deliveries that do not require resuscitation. The negative intrathoracic pressure generated by a vigorous, crying neonate potentially assists placental transfusion [21], estimated at 20–40 mL/kg [22], and increases oxygen delivery by increasing arterial oxygen content and cardiac output [23]. However, PPV does not appear to enhance placental transfusion [24]. It is also unclear if placental transfusion occurs in cord occlusion-induced perinatal asphyxia, especially in infants are born via c-section. In our study, including all available lambs, we were unable to demonstrate any advantage to neonatal blood volume in lambs with delayed cord clamping, with and without ventilation. However, asphyxial arrest causes cessation of cord blood

transfer. The non-arrested subgroup analysis showed reduced residual placental blood volume with delayed cord clamping, suggesting that placental transfusion does occur in this cohort.

We acknowledge several limitations of our study including obvious species differences. The use of maternal general anesthesia, fetal instrumentation and cesarean section could influence the results. However, these variables were common to all groups and between-group differences in results can be at least partly attributable to the randomized intervention. Unanticipated arrest was the biggest drawback of our model. As arrest occurred subsequent to the intervention period, we included them as an intent-to-treat analysis. The interventions may have contributed to progressive asphyxia and asystole. CC and epinephrine could affect post resuscitation hemodynamics, leading to large variation in results. Analyzing primary outcomes in the non-arrest subgroup was difficult due to small numbers, and these subgroup results are reported in the Supplementary Material (Table S1), where the current practice (ECC + V) resulted in a rapid return to baseline CBF. However, our unique translational model addresses important unanswered questions, with this study demonstrating complex interactions between ventilation techniques and cord clamp timing.

#### **5. Conclusions**

We conclude that higher airway pressures delivered by a 30 s SI breath in near-term asphyxiated lambs with DCC reduced both UV and PBF without affecting UA or CBF. A significant reduction in PBF with SI persists in the first few minutes after birth. We speculate that reduced PBF with SI may lead to delayed pulmonary vascular transition in this asphyxiated newborn model, already at increased risk for pulmonary hypertension/respiratory failure. However, DCC + SI resulted in lower CO2 and a higher PaO2/FiO2 ratio. On the basis of our findings, we suggest that SI as a ventilation strategy, especially in combination with DCC, should be used with caution in asphyxia. Our findings reinforce the need for randomized clinical trials to evaluate optimal cord management and ventilation strategies in asphyxiated newborns.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/children8050353/s1, Table S1: Subgroup without Arrest—Primary Outcomes.

**Author Contributions:** Conceptualization, J.N. and S.L.; methodology and acquisition of data J.N., L.D., S.G., C.K., J.H., L.N., S.K.B., D.S., V.A., P.C. and M.R.; data curation, C.K., J.H., S.G. and L.N.; writing—original draft preparation, J.N.; writing—review and editing, J.N. and S.L.; supervision, S.L.; project administration, J.N.; funding acquisition, J.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Institutes of Health—Eunice Kennedy Shriver National Institute of Child Health and Human Development, 1 Center, Bethesda, MD 20892 1 R03 HD086531-01 (J.N.) and American Academy of Pediatrics, Itasca, IL, 60143 Neonatal Resuscitation Program Research Grant 2017 (J.N.).

**Institutional Review Board Statement:** The study was approved by the Institutional Animal Care Committee (IACUC) at the University at Buffalo (Protocol PED10085N, IACUC ID AR202000013) and methods were consistent with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in this article.

**Conflicts of Interest:** The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

### **References**


**The Behaviour of Natural and Artificial Radionuclides in a River System: The Yenisei River, Russia as a Case Study The Behaviour of Natural and Artificial Radionuclides in a River System: The Yenisei River, Russia as a Case Study**

Lydia Bondareva, Valerii Rakitskii and Ivan Tananaev and Ivan Tananaev

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65743

Lydia Bondareva, Valerii Rakitskii

### **Abstract**

The Yenisei River is one of the largest rivers in the world. There is Mining and Chemical Combine (MCC) of Rosatom located at Krasnoyarsk, on the bank of the River Yenisei, 50 km downstream of the city of Krasnoyarsk. Since 1958 MCC used river's water for cooling of industrial nuclear reactors for the production of weapon plutonium—238Pu. Besides the pollution caused by industry-related radionuclides, pollution by natural radionuclide—uranium and its isotopes— are also investigated. Besides the natural uranium isotopes (234U, 235U, 238U), exclusive artificial isotope—236U was also found. Yenisei water was also polluted by high tritium content: from 4 Bq/L (back road value) to 200 Bq/L (some sample of water). The total amount of radionuclides investigated was about 20 radioisotopes. These radionuclides have different physical and chemical properties, different half-lives, and so on. Thus, the data on artificial radionuclides entering the Yenisei River water were obtained by long-term monitoring, which is likely to be connected with the activity of the industrial enterprises located on the river's banks of the studied area.

**Keywords:** Yenisei River, migration, radionuclide, Siberia, isotopes, Russia


**5. Conclusions**

In conclusion, our study enabled the selection of some RF that are able to predict with a certain good accuracy, the final nature of TIs discovered at 18F-FDG PET/CT imaging. Classic semiquantitative and volumetric PET/CT parameters did not reveal this ability. Furthermore, a good overlap in the extraction of RF between the two scanners was underlined.

**Author Contributions:** Conceptualization, F.D., R.G. (Roberto Gattaand) and D.A.; methodology, R.G. (Raffaele Giubbini) and N.P.; formal analysis, F.D. and R.G. (Roberto Gattaand); writing—original draft preparation, F.D., D.A. and R.G. (Raffaele Giubbini); writing—review and editing, N.P. and F.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Ethical review and approval were waived for this study due to the retrospective design of the study according to the local laws.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Data are not public, but are present in our institution.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**2.1 Safety of local anaesthetic**

Local anaesthetic agents are toxic at certain levels in systemic circulation. With any use of local anaesthetic, care must be taken to ensure LIA mixture is not injected directly into the patient's circulation. Local anaesthetic will inevitably be transported into circulation at a small concentration. However, it has been shown that the volume of local anaesthetic used in LIA technique is safe, and their levels in blood tests show sub-toxic concentration [18–20].

Fenten et al. took multiple plasma samples after LIA and torniquet release for TKA (at 20, 40, 60, 90, 120, 240, 360 minutes and 24 hours) [18]. The mix comprised of 200 mL 0.2% ropivacaine and 0.75 mg epinephrine 1:1000. Maximum ropivacaine concentration (total and unbound serum concentration) remained well below the assumed toxic threshold at all serum plasma samples. Stringer et al. studied LIA with the use of pain pump infusions for 48 hours post-operatively and measured ropivacaine concentration in THA and TKA patients [20]. The mixture contained 350-400 mg ropivacaine and defined their safe threshold concentration of 1–3 microg/mL intravascularly. Their intra-articular pain pump infusion commenced 12 hours post-operatively. Ropivacaine concentration was below 2 microg/ mL with LIA intraoperatively. Once intra-articular pain pump infusion commenced, peak ropivacaine concentration reached 0.65–4.36 microg/mL, however none of their patients experienced clinical signs of toxicity.


*Glyphosate Residues in Soil and Air: An Integrated Review DOI: http://dx.doi.org/10.5772/intechopen.93066*

*Pests, Weeds and Diseases in Agricultural Crop and Animal Husbandry Production*

[75]

**128**

**Year**

**Sample**

**Derivatization**

**Analytical method**

**LOD/LOQ μg/g**

**Other information**

**Reference**

**(pre- or**

**post-column)**

**preparation**

**(extraction/**

**cleanup)**

2008 2009 2009 2010 2011 2012 2013 2014 2014 2014

0.01 M

FMOC-CI

10% H PO3 4

0.6 M KOH

FMOC-CI

0.1

Μ

ΚΟΗ/SPE

FMOC-CI

0.2 M KOH

SWV using CFME

HPLC-ESI-MS/MS

using reversed-phase

C18

HPLC-FLD (λex = 267,

0.5 GLY

0.5 AMPA

λem =317 nm) using

reversed-phase C18

SPE-HPLC-ESI-MS/MS

0.37 GLY

Recoveries 85–126%

[61]

[78]

0.61 AMPA

0.6/2.0 ng/mL GLY

0.4/1.3 ng/mL

AMPA

(using HILIC)

SPE-HPLC-FLD (λex

=263 nm and λem

=317 nm)

25/83 μg/L GLY 0.02/0.05 GLY

0.01/0.03 AMPA

Water

1. Ca(ClO)2

2.OPA/ME

Water

2 M NH OH 4

1. CS2

UV 435 nm

1.1./3.7 μg/mL GLY

80–87%

2. Ammonical

solution of Cu(II)

CE/MS (negative with

20 GLY

Recoveries 76–121% (20/200/2000) for GLY

[74]

and 83–89% (40/400/4000) for AMPA

Pre-column conversion: 1. of glyphosate to glycine

by Ca(ClO)2; 2. followed by reaction with OPA/ME

in borate buffer (pH 9.5) to produce the fluorescent

1-(2′-hydroxyethylthio)-2-N-alkylisoindole

Recoveries 89–102%

Recoveries 79–117%

[76]

[77]

[57]

40 AMPA

0.08/0.25 mmol/L

GLY

amino capillary)

SIA-FLD (λex =270 nm,

λem =315 nm)

0.1 M KOH/CAX

cleaned up

2 M NH OH 4

TFAA/TFE

FMOC-CI

GC-NPD HPLC-FLD

0.01 GLY

0.025

Recoveries 88–94%

[72]

[55]

[73]

**Table 1.**

**131**

*Glyphosate Residues in Soil and Air: An Integrated Review*

derivative are formed suitable for GC analysis.

gradual degradation of GLY along with the presence of AMPA.

volatile derivatives for GC analysis. These derivatization approaches can be applied

GLY as a compound permits its detection (in conjunction with GC) by several detectors such as the flame photometric detector (FPD), flame ionization detector (FID), electron capture detector (ECD), nitrogen-phosphorus detector (NPD),and also the more sensitive and selective mass spectrometer detector (MSD). The quantification of GLY in the soil through NPD has reached an limit of quantification (LOQ ) equivalent of 0.02 mg/kg [93, 94]. GC analytical methods are reliable, sensitive, and selective, but the sample preparation is very time-consuming, complicated, and tedious as all ionic groups must be derivatized. In addition, they involve anhydrous conditions, extensive cleanup using solvent partitioning steps, charcoal elimination of pigments, and a large-volume anion of cation exchange.

Borjesson and Torstensson using GC-MS managed to sensitively detect GLY and AMPA in soil [48]. One point that should have been addressed was the content of humic acids a problem mentioned above as well. Extraction was tedious, involving extraction under basic conditions, adjustment of the pH to acidic, and then subjected to column purifications to achieve ligand-anion exchanges. To derivatize GLY and AMPA TFE and TFAA were used. By this way the respective ester and acetyl

Utilizing the S/N approach, they presented one of the lower LOQs of the bibliography for GLY, established at 0.006 mg/kg. The application of the method in soils collected from Swedish embankments after being treated with GLY revealed the

Bergstrom et al. investigated at laboratory level GLY and AMPA in sand and clay soils [13]. More specifically, its degradation was monitored using a GC-MS method, after derivatization with TFE and TFAA. The extraction of the soil was accomplished using an alkaline solution. Selected ion monitoring was utilized to enhance selectivity and optimize sensitivity of the method. The LOQ of the method was established at 0.01 mg/kg. The studied kinetics demonstrated that GLY had a very slow degradation rate in the clay soil. Concerning AMPA, though it is more tenacious than GLY (when derived from GLY), it degrades faster than GLY.

A 2019 study on GLY and AMPA analysis in soil showed that still GC-FPD can function as an adequate tool for such demanding analyses [89]. This research was intrigued by the obstacles observed in derivatization in connection with the by-product interferences in high-performance liquid chromatography (HPLC) analysis (when MS/MS mode is not utilized), which lead to inferior selectivity. Consequently, the soil samples were mixed, turned to powder, and then ultrasonic extracted using water assisted by a solid-phase extraction (SPE). After derivatization, the samples were subjected to chemical analysis. A breakthrough of this process was the three-cross derivatization, and the elaborate investigation of its optimization, aided by the orthogonal experimental design. Such design is fundamental in the selection of the optimum conditions, in this case, reaction temperature, time, and ratio of the derivatizing-coupling reagents.

GC-NPD was utilized by Hu and coworkers to analyze GLY in soil, using GC-MS for verification [72]. Extraction was performed in alkaline environment, followed

not only to soil (or sediment) but to other commodities as well [91].

*DOI: http://dx.doi.org/10.5772/intechopen.93066*

*<IP_ADDRESS> GC-MS*

*<IP_ADDRESS> GC-FPD*

*<IP_ADDRESS> GC-NPD*

*Main characteristics on the methods used for the determination of GLY and its main metabolite in soil matrices.*

#### *Glyphosate Residues in Soil and Air: An Integrated Review DOI: http://dx.doi.org/10.5772/intechopen.93066*

volatile derivatives for GC analysis. These derivatization approaches can be applied not only to soil (or sediment) but to other commodities as well [91].

GLY as a compound permits its detection (in conjunction with GC) by several detectors such as the flame photometric detector (FPD), flame ionization detector (FID), electron capture detector (ECD), nitrogen-phosphorus detector (NPD),and also the more sensitive and selective mass spectrometer detector (MSD). The quantification of GLY in the soil through NPD has reached an limit of quantification (LOQ ) equivalent of 0.02 mg/kg [93, 94]. GC analytical methods are reliable, sensitive, and selective, but the sample preparation is very time-consuming, complicated, and tedious as all ionic groups must be derivatized. In addition, they involve anhydrous conditions, extensive cleanup using solvent partitioning steps, charcoal elimination of pigments, and a large-volume anion of cation exchange.

#### *<IP_ADDRESS> GC-MS*

*Pests, Weeds and Diseases in Agricultural Crop and Animal Husbandry Production*

**130**

**Year**

**Sample**

**Derivatization**

**Analytical method**

**LOD/LOQ μg/g**

**Other information**

**Reference**

**(pre- or**

**post-column)**

1. Ca(ClO)2

SIC-FLD

0.03/0.10 GLY

Pre-column conversion: 1. of glyphosate to glycine

[90]

by Ca(ClO)2; 2. followed by reaction with OPA/ME

in borate buffer (pH 9.5) to produce the fluorescent

1-(2′-hydroxyethylthio)-2-N-alkylisoindole

*Solid-phase extraction = SPE; anion exchange = SAX; cation exchange = CAX; Flame photometric detector = FPD; nitrogen-phosphorus detector = NPD; fluorescence detector = FLD (λex = excitation;*

*λem = emission); capillary electrophoresis = CE; immunosensor = IS; square wave voltammetry = SWV; carbon-fiber microelectrode = CFME; double template imprinted polymer film-gold nanoparticlemodified pencil graphite electrode = DIPN-GNPs-PGE; carbon dot-labeled antibodies = lgG-CDs; sequential injection chromatography = SIC; sequential injection analysis = SIA; linker-assisted*

*enzyme-linked immunosorbent assay = L'ELISA; laser-induced fluorescence detection = LIF; Fluorenylmethylchloroformate = FMOC-Cl; 1-fluoro-2,4-dinitrobenzene = FDNB; trifluoroethanol = TFE;*

*N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide = MTBSTFA; 2,2,3,3,4,4,4-heptafluoro-1-butanol = HFB; trifluoroacetic anhydride = TFAA; heptafluorobutyric anhydride = HFBA; isopropyl*

*chloroformate = iso-PCF; o-phthalaldehyde/2-mercaptoethanol = OPA/ME; 4-toluenesulfonyl chloride = TsCl.*

*Main characteristics on the methods used for the determination of GLY and its main metabolite in soil matrices.*

**Table 1.**

2.OPA/ME

**preparation**

**(extraction/**

**cleanup)**

2019

Borjesson and Torstensson using GC-MS managed to sensitively detect GLY and AMPA in soil [48]. One point that should have been addressed was the content of humic acids a problem mentioned above as well. Extraction was tedious, involving extraction under basic conditions, adjustment of the pH to acidic, and then subjected to column purifications to achieve ligand-anion exchanges. To derivatize GLY and AMPA TFE and TFAA were used. By this way the respective ester and acetyl derivative are formed suitable for GC analysis.

Utilizing the S/N approach, they presented one of the lower LOQs of the bibliography for GLY, established at 0.006 mg/kg. The application of the method in soils collected from Swedish embankments after being treated with GLY revealed the gradual degradation of GLY along with the presence of AMPA.

Bergstrom et al. investigated at laboratory level GLY and AMPA in sand and clay soils [13]. More specifically, its degradation was monitored using a GC-MS method, after derivatization with TFE and TFAA. The extraction of the soil was accomplished using an alkaline solution. Selected ion monitoring was utilized to enhance selectivity and optimize sensitivity of the method. The LOQ of the method was established at 0.01 mg/kg. The studied kinetics demonstrated that GLY had a very slow degradation rate in the clay soil. Concerning AMPA, though it is more tenacious than GLY (when derived from GLY), it degrades faster than GLY.

#### *<IP_ADDRESS> GC-FPD*

A 2019 study on GLY and AMPA analysis in soil showed that still GC-FPD can function as an adequate tool for such demanding analyses [89]. This research was intrigued by the obstacles observed in derivatization in connection with the by-product interferences in high-performance liquid chromatography (HPLC) analysis (when MS/MS mode is not utilized), which lead to inferior selectivity. Consequently, the soil samples were mixed, turned to powder, and then ultrasonic extracted using water assisted by a solid-phase extraction (SPE). After derivatization, the samples were subjected to chemical analysis. A breakthrough of this process was the three-cross derivatization, and the elaborate investigation of its optimization, aided by the orthogonal experimental design. Such design is fundamental in the selection of the optimum conditions, in this case, reaction temperature, time, and ratio of the derivatizing-coupling reagents.

#### *<IP_ADDRESS> GC-NPD*

GC-NPD was utilized by Hu and coworkers to analyze GLY in soil, using GC-MS for verification [72]. Extraction was performed in alkaline environment, followed

by acidification in the dry extract. The authors stated that NH4OH was the most adequate extractant due to lesser extracted interferences than other alkaline agents, though in other works other agents are selected [48]. Derivatization was accomplished by the use of TFE and TFAA, followed by a liquid extraction using methylene chloride. The method verified the degradation of GLY in soil in apple orchards.

#### *2.3.4 Liquid chromatography and derivatization*

### *<IP_ADDRESS> Fundamentals*

The availability of derivatization techniques compatible with an aqueous extract or sample and the chromatographic separation makes LC a more attractive precolumn derivatization [91]. Derivatization approach is used to produce fluorescent derivatives and to enhance their retention in hydrophobic stationary phases prior to detection by fluorescence detection (FLD), UV detection, electrochemical detection (ECD), or tandem mass spectrometry (MS/MS). In post-column procedures, the most known reactions are ninhydrin derivatization accompanied by UV detection and fluorogenic labeling with o-phthalaldehyde (OPA) in mercaptoethanol or N,N-dimethyl-2-mercaptoethylamine after oxidation of glyphosate to glycine.

Although GLY and its derivatives show high sensitivity in LC determination, a laborious cleanup procedure such as ion-exchange column chromatography is required which may result in some sample loss and lower reproducibility, or many laboratories do not have the facilities required for this type of pre- or post-column fluorogenic labeling. The use of either hydrophilic/weak exchange or reversedphase/weak exchange mixed-mode chromatography without any derivatization, followed by diverse detection techniques including tandem mass spectrometry detection, is gaining interest [77, 95]. HPLC methods are highly sensitive with fluorogenic labeling, but they lack specificity and usually require a laborious cleanup procedure such as ion-exchange column chromatography, which may result in some sample loss and lower reproducibility.

#### *<IP_ADDRESS> Pre-column procedures*

#### *<IP_ADDRESS>.1 FMOC derivatization*

Pre-column procedures are a good alternative to post-column ones, and this has gradually come to play an important role in the analysis of glyphosate. The easier, less demanding and more current popular method to analyze these compounds is derivatization with 9-fluorenylmethylchloroformate (FMOC-Cl) followed by HPLC with FLD or MS/MS. A factorial experimental design was applied by a Chilean group in a critical analysis of this derivatization reaction [78]. The design was studied in aqueous soil extracts, unveiling the proper equilibrium between agents for the successful completion of the reaction. For example, excess of FMOC-Cl is required since there are also other active centers (amine-hydroxy groups) with which FMOC-Cl can react. Isotherm data verified the broad applicability of this method.

Back in the 1990s, Sancho et al. established a method for the analysis of GLY in soil samples that involved a pre-column derivatization step with FMOC-Cl and subsequent estimation by coupled-column liquid chromatography with fluorescence detection (LC–LC/FLD) [68]. However, for the determination of glyphosate in soils based on FMOC derivatization analytics, an extraction procedure including an SPE cleanup step has been used in many studies and considered more efficient [37]. In particular, Todorovic et al. extracted soils using sodium tetraborate. Once

**133**

*Glyphosate Residues in Soil and Air: An Integrated Review*

based on the analytical results on three different types of soils.

again, a group devoted substantial time in the extraction of GLY and AMPA from soil due to the complex sorption and desorption in soil which is also pH dependent. The sodium tetraborate performed better in terms of chromatographic efficiency than KOH extraction (more matrix interferences, more humic substances, etc.). The authors after derivatizing GLY and AMPA with FMOC-Cl cleaned up-enriched the extract with a polymeric SPE cartridge. Overall, the method was fit for purpose

Botero-Coy et al. have established a method based on LC–MS/MS, which was successfully applied to soil samples from Colombia and Argentina [77]. This work was an improvement of the previous work in the same domain [49]. In that work, the soil samples were extracted with potassium hydroxide solution and purified with SPE Oasis HLB cartridges. A pre-column derivatization step was also required in this method for which 9-fluorenylmethylchloroformate (FMOC-Cl) was used and the purification method using SPE cartridges was troublesome and expensive. Despite these difficulties, the analysis was conducted in Spanish soils with success. But, when soil samples from the mentioned countries were analyzed by the specific protocol, their high organic content proved an obstacle in the analysis. For this reason the authors introduced a dilution step of the extract assisted by pH adjustment to 9, before the SPE step. For SPE the polymeric reversed-phase Oasis HLB cartridges proved better in retaining-releasing the FMOC derivative than Oasis

Internal standard's use compensated possible downsides during sample preparation and corrected matrix effects. An additional tool in this work was the use of high-resolution mass spectrometry exploiting the time-of-flight technology. By this way additional interferences that would affect the analysis were further elucidated using the accurate mass full-acquisition data. It is noteworthy that the authors investigated MS ions used in the MS/MS mode. Interestingly, MRM transition containing the m/z 179 was problematic since it is related to FMOC and lacks specificity. In our work (see below), the specific ion was monitored only in AMPA transition, solving this issue [88]. Overall, the analysis verified the presence of GLY

Another work in the field of GLY analysis in soil/sludge using FMOC-Cl as a derivatizing agent was presented by Sun and coworkers [96]. In this context, an optimized sample preparation protocol was developed, applying extraction with sodium phosphate and trisodium citrate solutions (aqueous) and a purification step using hexane in acidified soil. The rationale behind the use of trisodium citrate was to counteract the effect of other metal ion complexing agents (such as Mg2+, Ca2+, etc.), in which GLY binds. The method was validated in three types of soils (and sludge samples) verifying that it was fit for purpose. The demonstrated LOQ was

A pre-column derivatization was applied by Druart and coworkers, embracing glufosinate also in their portfolio [60]. A detailed study was conducted on the parameters governing the extraction of the analytes from the matrix. Accelerated solvent extraction, ultrasonic extraction, and magnetic stirring agitation were tested to achieve optimum conditions. In the end agitation was selected. The group also optimized derivatization by selecting water as the solvent of the reaction, though the previous study showed that an equivalent mixture of H2O:ACN would compromise the solubility of both GLY and FMOC-Cl reagents [97]. In the same study, it was demonstrated that a C18 column of 30 cm superseded other columns

In addition our group has developed a methodology for GLY and AMPA detection in topsoils originating from Greece [88]. The sample preparation was envisaged by previous works (one of our group) [49, 98]. The LC–MS/MS method developed

tested, even a respective NH2 column broadly used for such separations.

*DOI: http://dx.doi.org/10.5772/intechopen.93066*

MAX used with good results.

and AMPA in the majority of samples.

determined at 0.04 mg/kg.

#### *Glyphosate Residues in Soil and Air: An Integrated Review DOI: http://dx.doi.org/10.5772/intechopen.93066*

*Pests, Weeds and Diseases in Agricultural Crop and Animal Husbandry Production*

*2.3.4 Liquid chromatography and derivatization*

sample loss and lower reproducibility.

*<IP_ADDRESS> Pre-column procedures*

*<IP_ADDRESS>.1 FMOC derivatization*

*<IP_ADDRESS> Fundamentals*

by acidification in the dry extract. The authors stated that NH4OH was the most adequate extractant due to lesser extracted interferences than other alkaline agents, though in other works other agents are selected [48]. Derivatization was accomplished by the use of TFE and TFAA, followed by a liquid extraction using methylene chloride. The method verified the degradation of GLY in soil in apple orchards.

The availability of derivatization techniques compatible with an aqueous extract

Pre-column procedures are a good alternative to post-column ones, and this has gradually come to play an important role in the analysis of glyphosate. The easier, less demanding and more current popular method to analyze these compounds is derivatization with 9-fluorenylmethylchloroformate (FMOC-Cl) followed by HPLC with FLD or MS/MS. A factorial experimental design was applied by a Chilean group in a critical analysis of this derivatization reaction [78]. The design was studied in aqueous soil extracts, unveiling the proper equilibrium between agents for the successful completion of the reaction. For example, excess of FMOC-Cl is required since there are also other active centers (amine-hydroxy groups) with which FMOC-Cl can react. Isotherm data verified the broad applicability of this

Back in the 1990s, Sancho et al. established a method for the analysis of GLY in soil samples that involved a pre-column derivatization step with FMOC-Cl and subsequent estimation by coupled-column liquid chromatography with fluorescence detection (LC–LC/FLD) [68]. However, for the determination of glyphosate in soils based on FMOC derivatization analytics, an extraction procedure including an SPE cleanup step has been used in many studies and considered more efficient [37]. In particular, Todorovic et al. extracted soils using sodium tetraborate. Once

or sample and the chromatographic separation makes LC a more attractive precolumn derivatization [91]. Derivatization approach is used to produce fluorescent derivatives and to enhance their retention in hydrophobic stationary phases prior to detection by fluorescence detection (FLD), UV detection, electrochemical detection (ECD), or tandem mass spectrometry (MS/MS). In post-column procedures, the most known reactions are ninhydrin derivatization accompanied by UV detection and fluorogenic labeling with o-phthalaldehyde (OPA) in mercaptoethanol or N,N-dimethyl-2-mercaptoethylamine after oxidation of glyphosate to glycine. Although GLY and its derivatives show high sensitivity in LC determination, a laborious cleanup procedure such as ion-exchange column chromatography is required which may result in some sample loss and lower reproducibility, or many laboratories do not have the facilities required for this type of pre- or post-column fluorogenic labeling. The use of either hydrophilic/weak exchange or reversedphase/weak exchange mixed-mode chromatography without any derivatization, followed by diverse detection techniques including tandem mass spectrometry detection, is gaining interest [77, 95]. HPLC methods are highly sensitive with fluorogenic labeling, but they lack specificity and usually require a laborious cleanup procedure such as ion-exchange column chromatography, which may result in some

**132**

method.

again, a group devoted substantial time in the extraction of GLY and AMPA from soil due to the complex sorption and desorption in soil which is also pH dependent. The sodium tetraborate performed better in terms of chromatographic efficiency than KOH extraction (more matrix interferences, more humic substances, etc.). The authors after derivatizing GLY and AMPA with FMOC-Cl cleaned up-enriched the extract with a polymeric SPE cartridge. Overall, the method was fit for purpose based on the analytical results on three different types of soils.

Botero-Coy et al. have established a method based on LC–MS/MS, which was successfully applied to soil samples from Colombia and Argentina [77]. This work was an improvement of the previous work in the same domain [49]. In that work, the soil samples were extracted with potassium hydroxide solution and purified with SPE Oasis HLB cartridges. A pre-column derivatization step was also required in this method for which 9-fluorenylmethylchloroformate (FMOC-Cl) was used and the purification method using SPE cartridges was troublesome and expensive. Despite these difficulties, the analysis was conducted in Spanish soils with success. But, when soil samples from the mentioned countries were analyzed by the specific protocol, their high organic content proved an obstacle in the analysis. For this reason the authors introduced a dilution step of the extract assisted by pH adjustment to 9, before the SPE step. For SPE the polymeric reversed-phase Oasis HLB cartridges proved better in retaining-releasing the FMOC derivative than Oasis MAX used with good results.

Internal standard's use compensated possible downsides during sample preparation and corrected matrix effects. An additional tool in this work was the use of high-resolution mass spectrometry exploiting the time-of-flight technology. By this way additional interferences that would affect the analysis were further elucidated using the accurate mass full-acquisition data. It is noteworthy that the authors investigated MS ions used in the MS/MS mode. Interestingly, MRM transition containing the m/z 179 was problematic since it is related to FMOC and lacks specificity. In our work (see below), the specific ion was monitored only in AMPA transition, solving this issue [88]. Overall, the analysis verified the presence of GLY and AMPA in the majority of samples.

Another work in the field of GLY analysis in soil/sludge using FMOC-Cl as a derivatizing agent was presented by Sun and coworkers [96]. In this context, an optimized sample preparation protocol was developed, applying extraction with sodium phosphate and trisodium citrate solutions (aqueous) and a purification step using hexane in acidified soil. The rationale behind the use of trisodium citrate was to counteract the effect of other metal ion complexing agents (such as Mg2+, Ca2+, etc.), in which GLY binds. The method was validated in three types of soils (and sludge samples) verifying that it was fit for purpose. The demonstrated LOQ was determined at 0.04 mg/kg.

A pre-column derivatization was applied by Druart and coworkers, embracing glufosinate also in their portfolio [60]. A detailed study was conducted on the parameters governing the extraction of the analytes from the matrix. Accelerated solvent extraction, ultrasonic extraction, and magnetic stirring agitation were tested to achieve optimum conditions. In the end agitation was selected. The group also optimized derivatization by selecting water as the solvent of the reaction, though the previous study showed that an equivalent mixture of H2O:ACN would compromise the solubility of both GLY and FMOC-Cl reagents [97]. In the same study, it was demonstrated that a C18 column of 30 cm superseded other columns tested, even a respective NH2 column broadly used for such separations.

In addition our group has developed a methodology for GLY and AMPA detection in topsoils originating from Greece [88]. The sample preparation was envisaged by previous works (one of our group) [49, 98]. The LC–MS/MS method developed

was adequate for the analysis of both active substances, showing that GLY and AMPA were detected in 37 and 45%, respectively, of the samples investigated. A breakthrough of this work was the association of the results with the land use utilizing geographical information system (GIS) databases.

GLY in soil is studied for registering not only its residual prevalence (including AMPA's) but also its degradation dynamics. With this in view, Zhang et al. investigated its dynamics using an HPLC-FD method, utilizing FMOC derivatization [3]. Results of this study showed that the degradation is dependent on the physicochemical parameters of the soil, exemplified by the pH. The behavior of GLY and AMPA was investigated in compost-amended soils by Erban and colleagues [86]. Soil depth was disclosed as a key factor on the concentrations detected. GLY and AMPA though showed a different behavior when moisture and saturated hydraulic conductivity are considered. More specifically, GLY was affected principally by moisture, whereas AMPA was impacted by this conductivity.